Method of screening antiobesity agents and animal model of obesity

ABSTRACT

A promoter for an angiopoietin-related growth factor (AGF), a vector comprising the promoter, a transformant comprising the promoter, and a method for screening an antiobesity agent, an antidiabetic agent, and/or a hypolipidemic agent by using the transformant, are disclosed. Further, an AGF knockout animal useful as an animal model for obesity, diabetes, and/or hyperlipemia is disclosed. Furthermore, an AGF transgenic animal useful for identification of a target molecule in a new drug development and/or a therapeutic agent for obesity, diabetes, and/or hyperlipemia is disclosed.

TECHNICAL FIELD

The present invention relates to a method of screening antiobesityagents, an animal model of obesity, and a promoter for anangiopoietin-related growth factor (hereinafter referred to as AGF).

BACKGROUND ART

Although the deleterious effect of obesity is widely known, there hasbeen a remarkable increase in obesity in recent years. It is well-knownthat obesity (i.e., overaccumulation of fat in fatty tissues) causesvarious diseases, and thus it is proposed that obesity should beaddressed as a disease to be treated. Diseases caused by obesityinclude, for example, lumbago, gonarthrosis, and osteoarthrosis. Suchorthopedic diseases are directly caused by a gain in body weight due toobesity. The overaccumulation of fat associated with obesity causesdiabetes, hyperlipemia, hypertension, or arteriosclerotic disease. Inparticular, it is known that an overaccumulation of visceral fat isinvolved in the development of such diseases (non-patent reference 1).

Basic methods for alleviating obesity include kinesitherapy and diettherapy, but to continue with such therapies is difficult. As methodsother than the kinesitherapy and diet therapy, medicaments are used. Atpresent, Sibutramine and orlistat are mainly used on a global scale.However, these medicaments have not only a weak, but also an adverseeffect. In Japan, only mazindol is authorized, but the applicationthereof is limited to severe obesity, and the period of administrationis also limited (non-patent reference 2).

Due to a modernization of society, the number of patients suffering fromdiabetes is rapidly increasing, not only in Japan but also globally. Inparticular, it is known that the development of type II diabetes havinga number of patients is involved in obesity or overaccumulation of fat.As with obesity, treatments for type II diabetes include kinesitherapyand diet therapy, but medicaments are used because it is difficult tocontinue these therapies. Patients suffering from severe diabetes aretreated with insulin, but the treatment with insulin has an adverseeffect such as hypoglycemia. As oral hypoglycemic drugs,thiazolidinediones or sulfonylurea agents are mainly used. However, thethiazolidinediones have an adverse effect such as hepatopathy, edema, orheart failure, and the SU agents have an adverse effect such as thepromotion of obesity, and thus, an agent for alleviating insulinresistance without an increase in body weight or such adverse effects isgreatly desired (non-patent reference 3).

It is considered that adipocytokines capable of promoting insulinresistance are produced and secreted from hypertrophied adipocytescontained in the visceral fat of an obese patient suffering fromdiabetes, and act on adipocytes and/or myocytes close to thehypertrophied adipocytes to promote insulin resistance. In adiposetissues of patients suffering from diabetes, adipocytes becomehypertrophied and change to tissues which are involved in the promotionof insulin resistance (non-patent references 4 and 5).

Leptin is well-known as a factor involved in the accumulation of adiposetissues which cause obesity or diabetes. Leptin is an inhibitory hormonefor bodyweight gain, and it is known that a deficiency of leptin causesobesity by promoting the appetite and reducing energy consumption. Thefindings of such factors involved in the accumulation of adipose tissuesand hypertrophy of adipocytes are very useful in developing therapeuticagents for diseases such as obesity, diabetes, or hyperlipemia(non-patent reference 6).

To identify a target molecule in a new drug development for obesity ordiabetes, or to develop a therapeutic agent therefor, it is important toprepare transgenic mice in which genes involved in the diseases aremodified, and to analyze the phenotypes thereof. A leptin-deficientmouse, an ob/ob mouse, becomes obese and shows insulin resistance,hyperinsulinemia, and a slight increase in blood glucose level. Aleptin-receptor-deficient mouse, a db/db mouse, shows obesity,hyperinsulinemia, hyperleptinemia, hyperinsulinemia, and severediabetes. These mice became obese and developed diabetes by thepromotion of feeding and a reduced energy consumption, due to thedeficiency of leptin action (non-patent reference 7). As a model mousefor obesity and type II diabetes, a KKA^(y) mouse is commonly used. TheKKA^(y) mouse shows obesity and a reduction in insulin sensibility anddevelops hyperglycemia, and thus, is used in the research anddevelopment of a therapeutic agent for diabetes. However, no genecausing the above phenotypes of the KKA^(y) mouse has been identified(non-patent reference 8). It is considered that many unknown genes areinvolved in obesity or diabetes (non-patent reference 9). Therefore, itis desirable to find such genes, prepare transgenic mice, and analyzethe phenotypes thereof, for developing medicaments for obesity ordiabetes and elucidating the diseases.

An angiopoietin-related growth factor (AGF) is a secretory proteinhaving a coiled-coil domain at the N-terminal side and a fibrinogen-likedomain at the C-terminal side. The AGF is identical with NL8 reported inpatent reference 1. It is reported that when CHO cells stably expressingNL8 are subcutaneously implanted into a nude mouse, the CHO cellsexhibit tumorigenicity. Sequences identical or homologous with the AGFare disclosed in patent references 2 to 16. The references disclose anexpression distribution thereof (patent references 4 and 5), an activityof inhibiting proliferation by stimulation of the vascular endothelialgrowth factor (VEGF) (patent reference 5), and a detection ofoverexpression thereof in human umbilical vein endothelial cell (HUVEC)(patent references 6 and 7), and further disclose that polypeptidesconsisting of amino acid sequences identical or homologous with the AGFare involved in angiogenesis, based on an expression in vascular tissuesor the like, tumorigenicity, and/or homology with family molecules.

Non-patent reference 10 discloses that, in a transgenic mouseoverexpressing the AGF in epidermal cells by using a K14 promoter,overangiogenesis occurred, microvessels under the skin were increased,and a proliferation of keratinocytes was activated. Non-patent reference11 discloses that the AGF exhibits an activity of proliferatingepidermal cells.

Because no AGF receptors were identified, tissues in which endogenousAGF functions and other physiological functions of AGF were unknown.Further, an antiobesity activity, an antidiabetic activity, or ahypolipidemic activity of AGF was unknown. Furthermore, an AGF promoterregion has not been reported.

[non-patent reference 1] Metabolism, (U.S.A.), 1987, vol. 36, p. 54-59

[non-patent reference 2] Nippon Rinsho, 2003, vol. 61, supplement 6,“Obesity”, p. 649-654

[non-patent reference 3] Nippon Rinsho, 2002, vol. 60, supplement 9,Shin-jidai no Tounyoubyougaku 3, p. 310-331

[non-patent reference 4] Igaku no ayumi, 2000, vol. 192, p. 513-518

[non-patent reference 5] Igaku no ayumi, 2000, vol. 192, p. 541-545

[non-patent reference 6] Trends in Molecular Medicine, (Netherlands),2002, vol. 8, no. 9, p. 442-447

[non-patent reference 7] K. G. M. M. Alberti, Paul Zimmet, and R. A.DeFronzo ed., C. J. Bailey, INTERNATIONAL TEXTBOOK OF DIABETES MELLITUS,2nd ed., (U.S.A.), John Wiley & Sons, Inc., 1997, p. 23.1-23.25

[non-patent reference 8] Nippon Rinsho, 2002, vol. 60, supplement 18, p.38-44

[non-patent reference 9] Saishin Igaku, 2002, vol. 57, supplement March,p. 536-544

[non-patent reference 10] Circulation, (U.S.A.), Nov. 5, 2002, vol. 106,no. 19, p. II-276-277

[non-patent reference 11] Two molecule of angiogenetic andanti-angiogenetic factor, —prevention of proliferation and metastasis ofcancer—, THE NIKKAN KOGYO SHINBUN LTD., Oct. 2, 2002, p. 5

[patent reference 1] International Publication No. WO99/15653 [patentreference 2] Japanese Unexamined Patent Publication (Kokai) No.2000-300263

[patent reference 3] Japanese Translation Publication (Kohyo) No.2001-517437

[patent reference 4] International Publication No. WO00/32221

[patent reference 5] International Publication No. WO00/53753

[patent reference 6] International Publication No. WO02/00690

[patent reference 7] International Publication No. WO02/08284

[patent reference 8] U.S. Patent Application Publication No.2003/0105011

[patent reference 9] U.S. Patent Application Publication No.2003/0105012

[patent reference 10] U.S. Patent Application Publication No.2003/0105013

[patent reference 11] U.S. Pat. No. 5,972,338

[patent reference 12] U.S. Pat. No. 6,057,435

[patent reference 13] U.S. Pat. No. 6,350,450

[patent reference 14] U.S. Pat. No. 6,413,770

[patent reference 15] U.S. Pat. No. 6,368,853

[patent reference 16] U.S. Pat. No. 6,420,542

DISCLOSURE OF INVENTION

Problems to be Solved by the Invention

An object of the present invention is to provide a method of screeningantiobestity agents and an animal model of obesity.

Means for Solving the Problems

The present inventors have conducted intensive studies, and, as aresult, can now reveal that substances capable of promoting AGF have anantiobesity activity, an antidiabetic activity, and/or a hypolipidemicactivity, by analyzing AGF knockout (KO) mice and AGF transgenic (Tg)mice. That is, the present inventors found that AGF knockout mice becomeremarkably obese, and that the AGF knockout mice are useful as an animalmodel of obesity (Example 4). Further, the present inventors found that,in the AGF knockout mice, there was an increase in weight of adiposetissues (Example 5), adipocytes were enlarged (Example 6), and there wasan increase in each amount of triglyceride contained in skeletal musclesor liver (Example 7). In contrast, the present inventors found in theAGF transgenic mice (i.e., mice in which AGF was overexpressed) that anincrease in body weight was suppressed (Example 4), that an increase inweight of adipose tissues was suppressed (Example 5), that anenlargement of adipocytes was suppressed (Example 6), and that eachamount of triglyceride contained in skeletal muscles or liver wasdecreased (Example 7). Furthermore, the AGF knockout mice developedsymptoms of diabetes in a glucose tolerance test (Example 8). From theabove findings, the present inventors found that substances capable ofpromoting AGF have an antiobesity activity, an antidiabetic activity,and/or a hypolipidemic activity.

In addition, the present inventors obtained various lengths of upstreamsequences of a human AGF gene, and have conducted intensive studies. Asa result, the present inventors found that a short sequence ofapproximately 300 bp upstream of the AGF gene unexpectedly exhibited apromoter activity, whereas longer sequences of approximately 400 bp toapproximately 3 kbp upstream of the AGF gene did not exhibit thepromoter activity. Further, the present inventors established a methodfor screening a substance capable of promoting an AGF expression byutilizing a DNA having the above promoter activity. From the abovefindings, the present inventors found that the method for screening asubstance capable of promoting an AGF expression is useful as a methodfor screening an antiobesity agent, an antidiabetic agent, and/or ahypolipidemic agent.

On the basis of the above findings, the present inventors provided anAGF knockout mouse, an AGF transgenic mouse, an AGF promoter, and amethod for screening a substance capable of promoting AGF, and thus thepresent invention was completed.

The present invention relates to:

[1] (a) a DNA exhibiting a promoter activity for an angiopoietin-relatedgrowth factor, and consisting of a nucleotide sequence in which 1 to 10nucleotides are substituted, deleted, added, and/or inserted in thenucleotide sequence consisting of nucleotides 2705-3001 of SEQ ID NO: 1,or

(b) a DNA exhibiting a promoter activity for an angiopoietin-relatedgrowth factor, and consisting of a nucleotide sequence having a 90% ormore homology with that consisting of nucleotides 2705-3001 of SEQ IDNO: 1,

[2] a DNA consisting of the nucleotide sequence consisting ofnucleotides 2705-3001 of SEQ ID NO: 1,

[3] a recombinant vector characterized by comprising the DNA of [1] or[2] and exhibiting a promoter activity for an angiopoietin-relatedgrowth factor, [4] a transformant characterized by comprising the DNA of[1] or [2] and exhibiting a promoter activity for anangiopoietin-related growth factor,

[5] a method for screening an antiobesity agent, an antidiabetic agent,and/or a hypolipidemic agent, characterized by comprising the steps of:

i) bringing a substance to be tested into contact with the transformantof [4], and

ii) measuring a promoter activity for an angiopoietin-related growthfactor and analyzing a test substance dependent change in the promoteractivity,

[6] the screening method of [5], wherein the transformant contains areporter gene located downstream of the DNA of [1] or [2], and thepromoter activity for an angiopoietin-related growth factor is measuredby analyzing an expression of the reporter gene,

[7] a nonhuman knockout animal characterized in that a polynucleotideencoding an angiopoietin-related growth factor is functionally deficienton a chromosome, and

[8] a nonhuman transgenic animal which is a nonhuman animal or anoffspring animal thereof obtained by ontogenesis from totipotent cellsin which a polynucleotide is introduced together with a CAG promoter,wherein the polynucleotide is carried on a chromosome, a polypeptideencoded by the polynucleotide is expressed in a somatic cell, and thepolypeptide is selected from the group consisting of:

(a) a polypeptide exhibiting an activity of suppressing an increase inbody weight, and comprising an amino acid sequence consisting of aminoacids 1-450 of SEQ ID NO: 3 or amino acids 1-433 of SEQ ID NO: 5,

(b) a polypeptide exhibiting an activity of suppressing an increase inbody weight, and comprising an amino acid sequence in which 1 to 10amino acids are substituted, deleted, and/or inserted in an amino acidsequence consisting of amino acids 1-450 of SEQ ID NO: 3 or amino acids1-433 of SEQ ID NO: 5,

(c) a polypeptide exhibiting an activity of suppressing an increase inbody weight, and encoded by a DNA which hybridizes under stringentconditions to a DNA encoding an amino acid sequence consisting of aminoacids 1-450 of SEQ ID NO: 3 or amino acids 1-433 of SEQ ID NO: 5, and

(d) a polypeptide exhibiting an activity of suppressing an increase inbody weight, and comprising an amino acid sequence having a 95% or morehomology with that consisting of amino acids 1-450 of SEQ ID NO: 3 oramino acids 1-433 of SEQ ID NO: 5.

Further, the present invention includes a method for preparing anonhuman transgenic animal (preferably a mouse) comprising the steps of:

introducing a DNA construct in which a CAG promoter is linked to theupstream of an AGF cDNA, into an embryonic stem (ES) cell,

selecting a clone in which the AGF is introduced,

introducing the selected clone into blastocysts,

transferring the manipulated eggs to a uterus of a

pseudopregnant female nonhuman mammal (preferably a mouse), and

breeding the nonhuman mammals (preferably mouse) and selecting, from theoffspring thereof, an animal having the DNA encoding the AGF on thegenome.

Furthermore, the present invention includes a method for preparing anonhuman knockout animal (preferably a mouse) comprising the steps of:

introducing a DNA construct in which a part of a genomic sequencecontaining ORF (Open Reading Frame) of the AGF gene is replaced with adrug resistant gene (such as a neomycin resistant gene), into an EScell,

culturing the ES cell in the presence of an appropriate drug (such asG418) to select a resistant strain,

introducing the resistant strain into blastocysts,

transferring the manipulated eggs to a uterus of a pseudopregnant femalenonhuman mammal (preferably a mouse), to obtain chimeric animals,

mating each chimeric animal (preferably a mouse) with a normal animal(preferably a mouse) to obtain heterozygous animals (preferably mice),and

mating the heterozygous animals with each other to obtain homozygousanimals (preferably mice).

The term “transgenic animal” as used herein means an animal in which apromoter and a gene are introduced into a chromosome to overexpress thegene at a desired location. The term “knockout animal” as used hereinmeans an animal in which an expression of a particular gene is deletedby gene manipulation of a chromosome.

The patent references 1 to 16 disclose polypeptides which may be used inpreparing the transgenic animal of the present invention and areidentical or homologous with a human or mouse AGF consisting of theamino acid sequence of SEQ ID NO: 3 or 5. These patent referencesdisclose that the polypeptides identical or homologous with the human ormouse AGF consisting of the amino acid sequence of SEQ ID NO: 3 or 5 areinvolved in angiogenesis, based on expressions in vascular tissues orthe like, tumorigenicity, and/or homologies with family molecules. Thepatent references 4 to 9 disclose many diseases including diabetes, butdisclose no support or evidence.

Further, the non-patent reference 10 discloses a functional analysis oftransgenic mice in which a K14 promoter was used to overexpress AGF inepidermal cells, and the non-patent reference 11 discloses that AGFexhibits an epidermal cell proliferating activity. WO03/083114 (patentreference 17), published after the priority date of the presentapplication, and Proceedings of the National Academy of Sciences of theUnited States of America, (U.S.A.), 2003, Vol. 100, p. 9494-9499(non-patent reference 12) disclose that transgenic mice in which AGF wasoverexpressed in epidermal cells utilizing a K14 promoter were used tofind that AGF exhibits an angiogenetic activity, an epidermal cellproliferating activity, a chondrocyte proliferating activity, anactivity of promoting wound healing, and a tissue generative activity.However, because no AGF receptors were identified, tissues in whichendogenous AGF functions and other physiological functions of AGF wereunknown. Further, an antiobesity activity or a hypolipidemic activity ofAGF was unknown.

A sequence of 129048 bp containing the sequence consisting ofnucleotides 2705-3001 of SEQ ID NO: 1 is registered as accession No.AC020931 in the Genbank database.

However, neither the use nor activities (including an AGF promoteractivity) of the sequence are disclosed in the database.

Although transgenic mice in which a K14 promoter was used foroverexpression in epidermal cells were reported (non-patent reference10, non-patent reference 12, and patent reference 17), there were noreports in which a transgenic mouse systemically expressing AGF and anAGF knockout mouse were generated and analyzed.

The present inventors first generated AGF transgenic mice systemicallyoverexpressing AGF and AGF knockout mice, and unexpectedly found thatthe AGF knockout mice are useful as an animal model for obesity,diabetes, and/or hyperlipemia, and that the AGF transgenic micesystemically overexpressing AGF are useful for identification of atarget molecule in a new drug development and a therapeutic agent forobesity, diabetes, and/or hyperlipemia.

Further, the present inventors first revealed that AGF or substancescapable of promoting AGF have an antiobesity activity, an antidiabeticactivity, and/or a hypolipidemic activity, by analyzing the AGF knockoutmice and the AGF transgenic mice systemically overexpressing AGF. Thepresent inventors first obtained an upstream sequence of the AGF genehaving an AGF promoter activity, and established a method for screeninga substance capable of promoting an AGF expression by utilizing theabove sequence. That is, the AGF knockout nonhuman animal, the AGFtransgenic nonhuman animal, the AGF promoter, and the method forscreening a substance capable of promoting AGF (i.e., an antiobesityagent, an antidiabetic agent, and/or a hypolipidemic agent) were firstprovided by the present inventors.

EFFECTS OF THE INVENTION

According to the AGF promoter of the present invention, a screeningsystem for an antiobesity agent, an antidiabetic agent, and/or ahypolipidemic agent can be provided.

A substance which is obtained by the screening method of the presentinvention and is capable of promoting an AGF expression is useful as anactive ingredient of an antiobesity agent, an antidiabetic agent, and/ora hypolipidemic agent.

The nonhuman AGF knockout animal of the present invention is useful asan animal model for obesity, diabetes, and/or hyperlipemia.

The nonhuman transgenic animal of the present invention may be used foridentification of a target molecule in a new drug development and/or atherapeutic agent for obesity, diabetes, and/or hyperlipemia.

BEST MODE FOR CARRYING OUT THE INVENTION

[1] DNA, Recombinant Vector, and Transformant of the Present Invention

The present inventors used a human genomic library to obtain partialsequences of approximately 200 bp, 300 bp, 400 bp, 600 bp, 800 bp, 1kbp, 1.3 kbp, and 3 kbp located upstream of the human AGF gene, anddetermined the nucleotide sequences thereof (nucleotide sequencesconsisting of nucleotides 2790-3001, nucleotides 2705-3001, nucleotides2604-3001, nucleotides 2406-3001, nucleotides 2206-3001, nucleotides2021-3001, nucleotides 1640-3001, and nucleotides 1-3001 of SEQ ID NO:1). Each DNA fragment was subcloned into plasmid pGV-B2 containing aluciferase gene as a reporter gene. It was confirmed whether theobtained upstream DNA regions exhibit a promoter activity by detectingan expression of the reporter gene in each fused plasmid (i.e.,luciferase activity). As a result, although the DNAs of approximately400 bp to 3 kbp did not exhibit the promoter activity, the shorter DNAof approximately 300 bp unexpectedly exhibited the promoter activity. Inthis connection, the further shorter DNA of approximately 200 bp did notexhibit the promoter activity.

The DNA of approximately 300 bp can be used to screen a substancecapable of regulating the AGF promoter activity, i.e., an antiobesityagent, an antidiabetic agent, and/or a hypolipidemic agent.

The DNA of the present invention includes, for example,

1) a DNA consisting of the nucleotide sequence consisting of nucleotides2705-3001 of SEQ ID NO: 1,

2) a DNA exhibiting a promoter activity for an angiopoietin-relatedgrowth factor, and consisting of a nucleotide sequence in which 1 to 10(preferably 1 to 7, more preferably 1 to 5, most preferably 1 to 3)nucleotides are substituted, deleted, added, and/or inserted in thenucleotide sequence consisting of nucleotides 2705-3001 of SEQ ID NO: 1,and

3) a DNA exhibiting a promoter activity for an angiopoietin-relatedgrowth factor, and consisting of a nucleotide sequence having a 90% ormore (preferably 95% or more, more preferably 97% or more) homology withthat consisting of nucleotides 2705-3001 of SEQ ID NO: 1.

The term “promoter activity for an angiopoietin-related growth factor(AGF)” as used herein means a promoter activity in the AGF gene,particularly a promoter activity of the DNA consisting of the nucleotidesequence consisting of nucleotides 2705-3001 of SEQ ID NO: 1. A methodfor judging whether or not a DNA of interest exhibits the “AGF promoteractivity” is not limited, but the promoter activity may be confirmed bya known conventional method. For example, an appropriate reporter geneDNA is linked at the 3′ downstream of a DNA to be judged, and the DNAconstruct is introduced into eukaryotic cells (preferably animal cellline). The cells are cultured, and an amount of the reporter geneexpressed in the cells is measured to confirm the AGF promoter activity.More particularly, the AGF promoter activity may be confirmed inaccordance with the method described in Example 12.

The DNA of the present invention may be prepared by, but is not limitedto, the following methods.

(1) Preparation Using PCR Method

A DNA consisting of the nucleotide sequence consisting of nucleotides2705-3001 of SEQ ID NO: 1 may be prepared by synthesizing a primer setof a primer consisting of a nucleotide sequence located at the 5′ sideof the sequence consisting of nucleotides 2705-3001 of SEQ ID NO: 1 anda primer consisting of a nucleotide sequence complementary to a sequencelocated at the 3′ side thereof (preferably a primer set of a primerconsisting of the nucleotide sequence of SEQ ID NO: 47 and a primerconsisting of the nucleotide sequence of SEQ ID NO: 46), and performinga PCR using these primers and a human genomic DNA. The human genomic DNAmay be prepared from an appropriate human tissue in accordance with aconventional method. A commercially available human genomic DNA may beused. More particularly, the DNA of the present invention may beprepared in accordance with, but is not limited to, the method describedin Example 11. Instead of the primers used in the method described inExample 11, other primers having restriction enzyme recognition sitesdifferent from those contained in the nucleotide sequences of SEQ IDNOS: 47 and 46, or other primers having lengths different from those ofthe nucleotide sequences of SEQ ID NOS: 47 and 46, may be used.

(2) Preparation Using DNA Synthesis

The DNA of the present invention may be prepared by chemicallysynthesizing the nucleotide sequence consisting of nucleotides 2705-3001of SEQ ID NO: 1 and the complementary strand, as several partial DNAfragments, and ligating these fragments with each other. The DNAfragments may be synthesized using a DNA synthesizer [for example, Oligo1000M DNA Synthesizer (Beckman) or 394 DNA/RNA Synthesizer (AppliedBiosystems)].

Those skilled in the art may prepare a DNA having the promoter activitysimilar to that of a naturally occurring promoter DNA, by performing amodification (for example, substitution, deletion, and/or addition) ofthe nucleotide sequence of the naturally occurring promoter DNA. The DNAof the present invention includes such a DNA having a nucleotidesequence in which one or more nucleotides are substituted, deleted,added, and/or inserted in the nucleotide sequence of a naturallyoccurring promoter DNA and exhibiting the promoter activity similar tothat of the naturally occurring promoter DNA. The nucleotidemodification may be carried out in accordance with, for example, amethod for introducing deletion by a restriction enzyme or DNAexonuclease, a method for introducing mutation by a site-directedmutagenesis [Nucleic Acid Res. 10, 6487 (1982)], a method for modifyinga promoter sequence by a PCR method using a mutated primer, or a methodfor directly introducing a synthetic mutated DNA [Maniatis, T. et al.(1989): “Molecular Cloning—A Laboratory Manual 2^(nd) Edit.” Cold SpringHarbor Laboratory, NY].

Whether or not the obtained DNA exhibits the promoter activity may beconfirmed by a known conventional method. For example, an appropriatereporter gene DNA is linked at the 3′ downstream of the obtained DNA,and the DNA construct is introduced into eukaryotic cells (preferablyanimal cell line). The cells are cultured, and an amount of the reportergene expressed in the cells is measured to confirm the promoteractivity. More particularly, the promoter activity may be confirmed inaccordance with the method described in Example 12.

The recombinant vector of the present invention may be prepared byintegrating the DNA of the present invention into a vector appropriatelyselected in accordance with a desired purpose. In the vector, a DNAcontaining a structural gene to be expressed may be inserted at the 3′downstream of the DNA of the present invention. The structural gene isnot particularly limited, so long as it encodes a protein. As thestructural gene, the whole or part of ORF (Open Reading Frame) may beused. For example, as described in Example 11, the recombinant vector ofthe present invention may be preferably prepared by integrating the DNAof the present invention (i.e., DNA having the AGF promoter activity)into a vector containing a reporter gene such as luciferase. The“reporter gene” is not particularly limited, so long as it is commonlyused. As the reporter gene, an enzyme gene in which a quantitativemeasurement may be easily performed is preferable. As the enzyme gene,there may be mentioned, for example, a chloramphenicol acetyltransferase gene (CAT) derived from a bacterial transposon, a luciferasegene (Luc) derived from a firefly, or a green fluorescent protein gene(GFP) derived from a jellyfish. The recombinant vector of the presentinvention may be preferably prepared in accordance with the methoddescribed in Example 11. Whether or not the obtained recombinant vectorexhibits the AGF promoter activity may be confirmed by a knownconventional method. For example, the recombinant vector is introducedinto eukaryotic cells (preferably animal cell line), the cells arecultured, and an amount of the reporter gene expressed in the cells ismeasured to confirm the promoter activity. More particularly, thepromoter activity may be confirmed in accordance with the methoddescribed in Example 12.

The transformant of the present invention may be prepared by introducingthe recombinant vector containing the DNA of the present invention intoa host cell appropriately selected in accordance with a desired purpose.For example, to construct a screening system for a substance capable ofregulating the hAGF promoter activity, a cell derived from mammals suchas a human, a mouse, or a rat (preferably a human-derived cell) may beused. As such a cell line, a cell having transcriptional regulatoryfactors or the like located in a commonly used cell may be used, andthere may be mentioned, for example, 293EBNA, HT-1080, or HepG2, whichare commercially available.

As a method for introducing a vector into a host cell, there may bementioned, for example, a DEAE-dextran method [Luthman, H. andMagnusson, G. (1983) Nucleic Acids Res., 11, 1295-1308], a calciumphosphate-DNA coprecipitation method [Graham, F. L. and van der Ed, A.J. (1973) Virology, 52, 456-457], a method using FuGENE6 (Nippon Roche),or an electroporation method [Neumann, E. et al. (1982) EMBO J., 1,841-845].

Whether or not the obtained transformant exhibits the AGF promoteractivity may be confirmed by measuring an amount of the reporter geneexpressed in the transformant. More particularly, it may be confirmed inaccordance with the method described in Example 12. A transformanthaving the AGF promoter activity may be selected by comparing it withthat transfected with a recombinant vector without the DNA of thepresent invention.

[2] Screening Method of the Present Invention

As shown in Examples 4 to 10, AGF exhibits an antiobesity activity, anantidiabetic activity, and/or a hypolipidemic activity. Therefore, asubstance useful as an antiobesity agent, an antidiabetic agent, and/ora hypolipidemic agent may be obtained by screening a substance capableof promoting the AGF promoter activity.

More particularly, an antiobesity agent, an antidiabetic agent, and/or ahypolipidemic agent may be screened by the screening method of thepresent invention, characterized by comprising the steps of:

i) bringing a substance to be tested into contact with the transformantof the present invention, and

ii) measuring the hAGF promoter activity, and analyzing (for example,detecting or measuring) a test substance dependent change in thepromoter activity.

Preferably, an antiobesity agent, an antidiabetic agent, and/or ahypolipidemic agent may be screened by the screening method of thepresent invention, characterized by comprising the steps of:

i) bringing a substance to be tested into contact with a celltransfected with a reporter gene fused to the DNA (preferably an hAGFpromoter region consisting of the nucleotide sequence consisting ofnucleotides 2705-3001 of SEQ ID NO: 1) of the present invention, and

ii) analyzing a test substance dependent change in the reporteractivity, in accordance with an expression of the reporter gene as anindex.

A reporter gene assay (Tamura et al., “Tensha inshi kenkyuhou”, YODOSHA,1993) is a method in which regulation of a gene expression is analyzed(for example, detected or measured) on the basis of an expression of areporter gene as a marker. A gene expression is generally regulated by apromoter region located at the 5′ upstream of the gene, and thus anamount of the gene expressed at the transcriptional stage may beestimated by measuring the promoter activity. When a test substanceactivates the promoter, the transcription of a reporter gene locateddownstream of the promoter region is activated. That is, the action ofactivating a promoter (i.e., the action of promoting an expression) maybe detected by replacing it with an expression of the reporter gene.Therefore, the action of a test substance on the regulation of AGFexpression may be detected by the reporter gene assay using the AGFpromoter region, on the basis of the expression of the reporter gene.The “reporter gene” fused to the DNA (preferably an hAGF promoter regionconsisting of the nucleotide sequence consisting of nucleotides2705-3001 of SEQ ID NO: 1) of the present invention is not particularlylimited, so long as it is commonly used. As the reporter gene, an enzymegene in which a quantitative measurement may be easily performed ispreferable. As the enzyme gene, there may be mentioned, for example, achloramphenicol acetyl transferase gene (CAT) derived from a bacterialtransposon, a luciferase gene (Luc) derived from a firefly, or a greenfluorescent protein gene (GFP) derived from a jellyfish. The reportergene may be functionally fused to the DNA (preferably an hAGF promoterregion consisting of the nucleotide sequence consisting of nucleotides2705-3001 of SEQ ID NO: 1) of the present invention. The reporter genefused to the DNA (preferably the hAGF promoter region) of the presentinvention is stably or transiently expressed in cells such as animalcells or yeast. An amount of the reporter gene expressed in thetransformants when a test substance is brought into contact therewithmay be compared to that when a test substance is not added, and a testsubstance dependent change in the promoter activity may be analyzed.

The transformant may be prepared in accordance with the methodsdescribed in the above [1]. A method for analyzing an amount of areporter gene expressed may be appropriately selected in accordance witha protein encoded by the reporter gene. For example, when the reportergene encodes a fluorescent protein such as luciferase, an amount of thereporter gene expressed may be determined by dissolving thetransformants by an appropriate method to obtain a cell lysate, addingluciferin as a substrate to a supernatant of the cell lysate, andmeasuring fluorescence by an appropriate fluorescence detector (forexample, ML3000; Dinatech laboratories). The reaction may be carried outusing a commercially available detection kit, for example, LuciferaseAssay System (Promega).

A substance capable of promoting the AGF expression, i.e., anantiobesity agent, an antidiabetic agent, and/or a hypolipidemic agent,may be screened by carrying out the above steps. More particularly, themethod described in Example 12 is preferable as the screening method. Asthe substance capable of promoting the promoter activity, it ispreferable to select a substance in which the activity of activating thepromoter is 1.5 times or more with respect to that in the absence of thesubstance.

Substances to be tested which may be used in the screening method of thepresent invention are not particularly limited, but there may bementioned, for example, commercially available compounds (includingpeptides), various known compounds (including peptides) registered inchemical files, compounds obtained by combinatorial chemistry techniques(Terrett et al., J. Steele. Tetrahedron, 51, 8135-8137, 1995), culturesupernatants of microorganisms, natural components derived from plantsor marine organisms, animal tissue extracts, or compounds (includingpeptides) obtained by chemically or biologically modifying compounds(including peptides) selected by the screening method of the presentinvention.

[3] The Nonhuman Knockout Animal and Nonhuman Transgenic Animal of thePresent Invention

The nonhuman knockout animal of the present invention is notparticularly limited, so long as a polynucleotide encoding AGF isfunctionally deficient on a chromosome. The nonhuman knockout animal maybe prepared in accordance with a method commonly used in preparing aknockout animal [for example, see “Saisin doubutsu saibou jikkenmanual”, published by LIC, Chapter 7, p. 361-408 (1990)], by utilizing agenomic sequence containing ORF of the AGF gene or a genomic sequencecontaining the upstream and/or downstream sequences of the AGF gene. Thegenomic sequence may be selected in accordance with an animal (species)to be used. For example, when a mouse is used, the sequence of accessionNo. AC073775.2 in GenBank may be utilized. Alternatively, the AGFknockout animal may be obtained by selecting a mouse functionallydeficient in the AGF gene from mice prepared by a random mutagenesismethod (for example, see Nature, 392, 608-611, 1998).

More particularly, the nonhuman knockout animal of the present inventionmay be prepared in accordance with, for example, the method described inExample 1. That is, a genomic sequence containing ORF of the AGF gene isused to prepare a DNA construct in which a part thereof is replaced witha drug resistant gene (such as a neomycin resistant gene). ES cells aretransfected with the DNA construct, and cultured in the presence of anappropriate drug (such as G418) to obtain resistant strains. Theresistant strains are analyzed by, for example, Southern blotting, toselect clones in which a desired homologous recombination occurs. Eachclone is microinjected into a blastocyst, and the manipulated eggs aretransferred to a uterus, to obtain chimeric mice. Each chimeric mouse ismated with a normal mouse to obtain heterozygous mice. Further, theheterozygous mice are mated with each other to obtain homozygous mice inaccordance with the Mendel's laws.

Methods not utilizing ES cells, such as a method in which a mixturecontaining a gene of interest and eggs is treated with calciumphosphate, a method in which a gene is directly introduced into anucleus in fertilized eggs at the pronuclear stage under aphase-contrast microscope (a microinjection method; U.S. Pat. No.4,873,191), a method in which eggs are infected with a retroviral vectorcontaining a gene, or a sperm vector method in which a gene isintroduced into eggs via sperm [M. Lavitrano et al., Cell 57(5): 717-723(1989)], are known, and may be used in preparing the nonhuman knockoutanimal (or nonhuman transgenic animal described below) of the presentinvention.

The nonhuman knockout animal (or nonhuman transgenic animal describedbelow) of the present invention may be prepared by utilizing anyvertebrates other than a human.

Various vertebrates, such as a mouse, rat, rabbit, miniature pig, goat,sheep, or cattle, were used to generate knockout animals in whichvarious genes were incorporated or an expression level was modified.Such species are included in the nonhuman knockout animal of the presentinvention. As the nonhuman knockout animal (or nonhuman transgenicanimal described below) of the present invention, rodents arepreferable, and a mouse is most preferable.

As described in Examples, the nonhuman knockout animal of the presentinvention becomes remarkably obese (Example 4).

Further, in the nonhuman knockout animal of the present invention, thereis an increase in weight of adipose tissues (Example 5), adipocytes areenlarged (Example 6), there is an increase in each amount oftriglyceride contained in skeletal muscles or liver (Example 7), andsymptoms of diabetes are developed in a glucose tolerance test (Example8). These phenotypes resemble symptoms in obese subjects or patientssuffering from diabetes or hyperlipemia, and thus the nonhuman knockoutanimal of the present invention is useful as an animal model forobesity, diabetes, and/or hyperlipemia. That is, the nonhuman knockoutanimal of the present invention can be used not only in screeningmedicaments for treating or preventing obesity, diabetes, and/orhyperlipemia, but also in elucidating the mechanisms of such diseasesand for a safety test of the medicament screened. The present inventionincludes the use of the nonhuman AGF knockout animal as an animal modelfor obesity, diabetes, and/or hyperlipemia.

The nonhuman transgenic animal of the present invention may be preparedin accordance with the above procedures described with respect to thenonhuman knockout animal of the present invention, except that apolynucleotide which encodes a polypeptide (hereinafter referred to as“polypeptide for preparing a transgenic animal”) selected from the groupconsisting of:

(a) a polypeptide exhibiting an activity of suppressing an increase inbody weight, and comprising an amino acid sequence consisting of aminoacids 1-450 of SEQ ID NO: 3 or amino acids 1-433 of SEQ ID NO: 5,

(b) a polypeptide exhibiting an activity of suppressing an increase inbody weight, and comprising an amino acid sequence in which 1 to 10amino acids are substituted, deleted, and/or inserted in an amino acidsequence consisting of amino acids 1-450 of SEQ ID NO: 3 or amino acids1-433 of SEQ ID NO: 5,

(c) a polypeptide exhibiting an activity of suppressing an increase inbody weight, and encoded by a DNA which hybridizes under stringentconditions to a DNA encoding an amino acid sequence consisting of aminoacids 1-450 of SEQ ID NO: 3 or amino acids 1-433 of SEQ ID NO: 5, and

(d) a polypeptide exhibiting an activity of suppressing an increase inbody weight, and comprising an amino acid sequence having a 95% or morehomology with that consisting of amino acids 1-450 of SEQ ID NO: 3 oramino acids 1-433 of SEQ ID NO:

and is linked to a CAG (modified chicken beta-actin promoter with CMV-IEenhancer) promoter [GENE, 108(1991) 193-200] is used as a gene to beintroduced.

As the polypeptide for preparing a transgenic animal, a human AGFconsisting of the amino acid sequence consisting of amino acids 1-450 ofSEQ ID NO: 3, or a mouse AGF consisting of the amino acid sequenceconsisting of amino acids 1-433 of SEQ ID NO: 5 is preferable.

The amino acid sequence of SEQ ID NO: 3 is that of a human AGFprecursor. The human AGF has a signal sequence (−20 to −1) at the Nterminus thereof, and the signal sequence is cleaved when the precursoris secreted to the outside of cells. A human matured AGF, which isgenerated by cleaving the signal sequence and is composed of the aminoacid sequence consisting of amino acids 1-450 of SEQ ID NO: 3, hasphysiological activities.

Similarly, the amino acid sequence of SEQ ID NO: 5 is that of a mouseAGF precursor. The mouse AGF has a signal sequence (−24 to −1) at the Nterminus thereof, and the signal sequence is cleaved when the precursoris secreted to the outside of cells. A mouse matured AGF, which isgenerated by cleaving the signal sequence and is composed of the aminoacid sequence consisting of amino acids 1-433 of SEQ ID NO: 5, hasphysiological activities.

As the above polypeptide (b) which may be used as the polypeptide forpreparing a transgenic animal [i.e., a polypeptide exhibiting anactivity of suppressing an increase in body weight, and comprising anamino acid sequence in which 1 to 10 (for example, one to several) aminoacids are substituted, deleted, and/or inserted in an amino acidsequence consisting of amino acids 1-450 of SEQ ID NO: 3 or amino acids1-433 of SEQ ID NO: 5], there may be mentioned, for example, apolypeptide exhibiting an activity of suppressing an increase in bodyweight, and comprising an amino acid sequence in which preferably 1 to7, more preferably 1 to 5 amino acids are substituted, deleted, and/orinserted in an amino acid sequence consisting of amino acids 1-450 ofSEQ ID NO: 3 or amino acids 1-433 of SEQ ID NO: 5.

To maintain the functions of the original polypeptide, the amino acid tobe substituted is preferably an amino acid having properties similar tothose of the original amino acid. For example, amino acids belonging toeach of the following groups have properties similar to those of othermembers in the group. When these amino acids are substituted with otheramino acids in the same group, the essential functions of the originalprotein are often maintained. Such amino acid substitution is called aconservative substitution, and is known as a method for changing anamino acid sequence while maintaining the polypeptide functions.Nonpolar amino acids: Ala, Val, Leu, Ile, Pro, Met, Phe, and TrpUncharged amino acids: Gly, Ser, Thr, Cys, Tyr, Asn, and Gln Acidicamino acids: Asp and Glu Basic amino acids: Lys, Arg, and His

A method for judging whether or not a polypeptide of interest exhibitsthe activity of suppressing an increase in body weight is not limited,but it may be confirmed by, for example, the method described in Example4. That is, the activity may be confirmed by breeding transgenicanimals, prepared by utilizing a gene encoding the polypeptide, with anormal diet or a high fat diet, and comparing changes in body weightwith those of wild-type animals.

With respect to the above polypeptide (c) which may be used as thepolypeptide for preparing a transgenic animal, the “stringentconditions” include, as hybridization conditions, conditions of “5×SSPE,5× Denhard's solution, 0.5% sodium dodecyl sulfate (SDS), 40% formamide,and 200 μg/mL salmon sperm DNA, at 37° C. overnight”, and, as morestringent hybridization conditions, conditions of “5×SSPE, 5× Denhard'ssolution, 0.5% SDS, 50% formamide, and 200 μg/mL salmon sperm DNA, at42° C. overnight”. Further, washing conditions include mild conditionssuch as “5×SSC and 1% SDS, at 42° C.”, usual conditions such as “0.5×SSCand 0.1% SDS, at 42° C.”, and more stringent conditions such as “0.2×SSCand 0.1% SDS, at 65° C.”. The “5×SSPE” contains 50 mmol/L sodiumphosphate (pH 7.4), 0.75 mol/L NaCl, and 5 mmol/L EDTA. The “5×SSC”contains 0.75 mol/L NaCl and 75 mmol/L sodium citrate (pH 7.0).

The homology in the above polypeptide (d) which may be used as thepolypeptide for preparing a transgenic animal is at least 95% or more,preferably 97% or more. The homology between amino acid sequences may becalculated by a BLAST search algorithm. More particularly, it may becalculated using a b12seq program (Tatiana A. Tatusova and Thomas L.

Madden, FEMS Microbiol. Lett., 174, 247-250, 1999) in a BLAST package(sgi32 bit edition, version 2.0.12; obtained from NCBI) in accordancewith a default parameter. As a pairwise alignment parameter, a program“blastp” is used. Further, “0” as a Gap insertion cost value, “0” as aGap elongation cost value, “SEG” as a filter for a Query sequence, and“BLOSUM62” as a Matrix are used, respectively.

As the polypeptide for preparing a transgenic animal, a polypeptide inwhich a signal sequence is added to the N terminus of any one of thepolypeptides (a) to (d) is preferable. The signal sequence is notparticularly limited, so long as it may lead a polypeptide to passthrough the membrane. As the signal sequence, signal sequences describedin Biochemistry, 28(3), 923-930, 1989 may be used. As the polypeptidefor preparing a transgenic animal, a polypeptide in which a signalsequence (−20 to −1) in the amino acid sequence of SEQ ID NO: 3 or asignal sequence (−24 to −1) in the amino acid sequence of SEQ ID NO: 5is added to the N terminus of any one of the polypeptides (a) to (d) ismore preferable, and a polypeptide consisting of the amino acid sequenceof SEQ ID NO: 3 or 5 is most preferable.

The origin of the polypeptide for preparing a transgenic animal is notlimited to a human or a mouse. For example, a polypeptide derived fromorganisms other than a human or a mouse, or a polypeptide obtained,using genetic engineering techniques, by artificially modifying an aminoacid sequence consisting of amino acids 1-450 of SEQ ID NO: 3 or aminoacids 1-433 of SEQ ID NO: 5, may be used, so long as it is included inany one of the polypeptides (a) to (d).

The polynucleotides encoding the polypeptides (a) to (d), i.e., thepolynucleotides which may be used in preparing the nonhuman transgenicanimal of the present invention, include DNAs and RNAs, and DNAs arepreferable. As the polynucleotide, there may be mentioned, for example,a polynucleotide which encodes a polypeptide having an activity ofsuppressing an increase in body weight and contains a nucleotidesequence consisting of nucleotides 61-1410 of SEQ ID NO: 2 ornucleotides 73-1371 of SEQ ID NO: 4. A polynucleotide consisting of anucleotide sequence of SEQ ID NO: 2 or 4, or a polynucleotide consistingof a nucleotide sequence consisting of nucleotides 61-1410 of SEQ ID NO:2 or nucleotides 73-1371 of SEQ ID NO: 4, is preferable.

The nonhuman transgenic animal of the present invention may be prepared,for example, by the method described in Example 2. That is, a CAGpromoter is linked to the upstream of an AGF cDNA, to prepare a DNAconstruct. The obtained DNA construct is introduced into ES cells, andclones expressing AGF are selected by, for example, a Western blottinganalysis. Each clone is microinjected into a blastocyst, and themanipulated eggs are transferred to a uterus, to obtain chimeric mice.Each chimeric mouse is mated with a normal mouse to obtain transgenicmice.

The nonhuman transgenic animal of the present invention can be used foridentification of a target molecule in a new drug development and/or atherapeutic agent for obesity, diabetes, and/or hyperlipemia. Forexample, the nonhuman transgenic animal of the present invention (suchas an AGF transgenic mouse) is mated with an animal (such as a mouse) inwhich a gene X of interest is modified, to obtain offspring in whichboth genes are modified. The phenotypes of the offspring may beanalyzed. When the offspring in which both genes are modified do notexhibit an antiobesity activity, an antidiabetic activity, and/or ahypolipidemic activity, it is revealed that the gene X has anantiobesity activity, an antidiabetic activity, and/or a hypolipidemicactivity. Further, it is revealed that the gene X and/or an agent forpromoting the function of the gene X may be used as a therapeutic agentfor obesity, diabetes, and/or hyperlipemia.

Further, a gene whose expression is changed in the nonhuman transgenicanimal of the present invention is considered to be a target molecule ina new drug development for obesity, diabetes, and/or hyperlipemia, andthus the gene, or agonists or antagonists thereof may be used as anantiobesity agent, an antidiabetic agent, and/or a hypolipidemic agent.The nonhuman transgenic animal of the present invention is useful inproviding materials (such as tissues or blood) for identification of atarget molecule in a new drug development.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples. In this connection, thefollowing procedures may be performed in accordance with known methods(for example, “Molecular Cloning”, Sambrook, J. et al., Cold SpringHarbor Laboratory Press, 1989), unless otherwise specified. Further,when a commercially available reagent or kit is used, procedures may beperformed in accordance with a protocol attached thereto.

Knockout animals and transgenic animals may be prepared in accordancewith “Manipulating the Mouse Embryo. A Laboratory Manual.” 2nd Edition,B. Hogan, R. Beddington, F. Costantini, E. Lacy, Cold Spring Harbor,N.Y., Cold Spring Harbor Laboratory Press, 1994, unless otherwisespecified. Further, chimeric mice may be prepared by using ES cells inaccordance with A L Joyner: “Gene Targeting, A Practical Approach”,OXFORD UNIVERSITY PRESS, 1993; or Shinich Aizawa, Biomanual series 8,“Gene Targeting, ES saibou wo mochiita henimausu no sakusei”, Youdosha,1994, unless otherwise specified.

Example 1 Preparation of AGF KO Mice

(1) Construction of Targeting Vector

A targeting vector containing a genomic sequence (5′ long arm) at the 5′side of the mouse AGF gene, a pgk promoter, a neomycin resistant gene, agenomic sequence (3′ short arm) containing a part of exon 2 and thewhole of exon 3 in the mouse AGF gene, and an HSV-tk gene, in thisorder, was prepared in accordance with the following procedures.

A cDNA corresponding to the full-length of the coding region of mouseAGF protein was prepared by the procedures described in Example 1 ofWO03/083114. The cDNA was used as a probe to screen a mouse genomiclibrary (Mouse Genomic, 129 SVJ Library; Stratagene) in accordance witha manual attached thereto. A phage clone containing a sequence ofapproximately 17.9 kbp (corresponding to 90644 to 108544 inmouse-pub-genome sequence AC073775.2 containing the AGF gene) wasisolated and subcloned into plasmid pBluescript (Stratagene). Theobtained plasmid clone (pBN2) was digested with restriction enzymes SalIand MfeI to obtain the 5′ long arm of approximately 6.2 kbp (containing90664 to 96900 in mouse-pub-genome sequence AC073775.2). Further, a PCRwas carried out using the plasmid pBN2 as a template, together with aprimer set [SEQ ID NO: 6 (CTAGACTAGTTGCAAAGGCGTGCGGCGG; artificialsequence) and SEQ ID NO: 7 (CTAGACTAGTGGATCCGCAGGCTTGCTTTGACTTAC;artificial sequence)] to obtain the 3′ short arm of approximately 2.0kbp (containing 105903 to 107914 in mouse-pub-genome sequenceAC073775.2). The 5′ long arm and the 3′ short arm were inserted into theXhoI site and the XbaI site of plasmid PPNT [Cell, 1991, 65(7),1153-1163], respectively, to construct the targeting vector.

(2) Preparation of Homologous Recombinant ES Cells

The obtained targeting vector was digested with restriction enzyme NotI,and introduced into ES cell line R1 (Proceedings of the National Academyof Sciences, Vol 90, 8424-8428, 1993) by electroporation. The ES cellswere cultured in a medium containing G418 to obtain resistant strains.DNAs were extracted from ES cells, and clones in which only a desiredhomologous recombination occurred were identified by Southern blotting.More particularly, each DNA was digested with restriction enzymeHindIII, and analyzed by Southern blotting using, as a probe, a DNAconsisting of the nucleotide sequence of SEQ ID NO: 8(GCCCATGGAGGGATTGTGCAGAGGCTCACGGGGCAGGTCACTGGCAGAGTGGAGTGTATGACCTGCGGCTGGGCCGTCGTGTAGTAGCCGTGTGGTGTGAACAGCAGCAGGAAGTGGAGGCTGGACTGTCATCCAGAGACGGCAGGACGGCTCTGTCAACTTCTTCACCAACTGGCAGCACTACAAGGTGTGTGCTTGTGGTGGGGGTGTCAGAGACTGCTGGGCAGAGAGGACGCCCCCACCCTCTTCCTCCTACCCTTCCAGGCGGGCTTTGGGCGTCCAGAAGGAGAATACTGGCTGGGCCTGGAACCTGTGCATCAGGTGACAAGCCGTGGGGACCACGAGCTGCTGATACTCCTAGAGGACTGGGGGGGCCGTGCAGCACGCGCCCACTACGACAGCTTCTCCTTGGAGCCTGAGAGTGACCACTACCGTCTGCGGCTTGGCCAGTACCACGGCGATGCCGGAGACTCCCTCTCTTGGCACAATGACAAAACCTTTCAGCACTGTGGATAGGGACAGAGACTCATATTCTG; mouse) containing exon 4 andexon 5 in AGF. As a result, a DNA fragment of 6.5 Kb was detected inhomologous recombinant clones, in comparison with that of 4.6 Kb in thewild-type.

(3) Preparation of AGF KO Mice

The obtained ES cell line was microinjected into blastocysts preparedfrom BDF2 mice which were F2 hybrid mice of C57BL/6 and DBA/2 mice, andthe manipulated eggs were transferred to a uterus, to obtain chimericmice from the pregnant mice. The chimeric mice were mated with C57BL/6mice to obtain heterozygous mice (hereinafter referred to as AGFheterozygous KO mice) having a mutated allele lacking in the initiationcodon of AGF. The AGF heterozygous KO mice were mated with each other toobtain homozygous mice (hereinafter referred to as AGF homozygous KOmice). A PCR using genomic DNA isolated from the tail of each offspringmouse as a template was carried out to confirm the genotype thereof fromthe size of each DNA fragment obtained by the PCR, as described below.That is, the tail was treated with proteinase K, and a phenol/chloroformextraction was carried out to obtain DNA. The extracted DNA wascollected by an isopropanol precipitation followed by an ethanolprecipitation, and dissolved in a Tris-EDTA buffer (hereinafter referredto as TE solution). The following primers were designed on the basis ofa sequence of the neomycin resistant gene and a genomic sequence to bedeleted by targeting: (neomycin resistant gene) Forward primer: SEQ IDNO: 9 (5′-agaggctattcggctatgac-3′; artificial sequence) Reverse primer:SEQ ID NO: 10 (5′-caccatgatattcggcaagc-3′; artificial sequence) (genomicDNA) Forward primer: SEQ ID NO: 11 (5′-tggcctctgttatcatgctc-3′; mouse)Reverse primer: SEQ ID NO: 12 (5′-ctacctacatccactcctac-3′; mouse)

The genomic DNA obtained from each offspring mouse was used togetherwith the above primers and a DNA polymerase (ExTaq; Takara) to performPCRs. In the PCRs, a thermal denature at 95° C. for 5 minutes wascarried out, a cycle composed of reactions at 95° C. for a minute, at60° C. for a minute, and at 72° C. for a minute was repeated 30 times,and an elongation reaction at 72° C. for 7 minutes was carried out. Thesizes of fragments amplified by the PCRs were analyzed. When anoffspring mouse has the mutated allele, a band of 545 bp is detected inthe PCR for detecting the neomycin resistant gene. When an offspringmouse has the wild-type allele, a band of 322 bp is detected in the PCRfor detecting the genomic DNA containing mouse AGF exon 1. The genotypeof each mouse was determined from the results. As a result, in the AGFhomozygous KO mice, the band of the mutated allele was detected, but theband of the wild-type allele was not detected. In the AGF heterozygousKO mice, the band of the mutated allele and that of the wild-type allelewere detected. In the wild-type mice (hereinafter referred to aslittermate WT mice), the band of the mutated allele was not detected,but the band of the wild-type allele was detected.

Further, the genotype of each mouse was analyzed by Southern blottingdescribed in Example 1(2). As a result, in the AGF homozygous KO mice, aband of 6.5 kbp derived from the mutated allele was detected. In the AGFheterozygous KO mice, a band of 6.5 kbp derived from the mutated alleleand that of 4.6 kbp derived from the wild-type allele were detected. Inthe littermate WT mice, the band of 4.6 kbp derived from the wild-typeallele was detected.

Furthermore, each blood sample collected from the AGF KO mice wasallowed to stand at 37° C. for 30 minutes, and centrifuged to obtain aserum as the supernatant. The serum was diluted to 1/20 with a lysisbuffer [0.5 mol/L HEPES (pH7.2), 1% Triton X-100, 10% glycerol, 10mmol/L Na₄P₂O₇, 0.1 mol/L NaF, 0.1 mmol/L Na₃VO₄, 4 mmol/L EDTA (pH 8),0.05 mg/mL aprotinin, 1 mmol/L PMSF, 0.1 mmol/L leupeptin, and 0.025mmol/L Pepstatin A], and further diluted with an equal volume of a 2×SDSsample buffer. Each sample (20 μL per lane) was subjected to 10%acrylamide gel electrophoresis, followed by Western blotting. In theWestern blotting, TBS-T [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl,and 0.05% (w/v) Tween 20] containing 5% bovine serum albumin (BSA) wasused as a blocking agent, an anti mouse AGF antibody (WO03/083114) wasused as the first antibody, and an anti rabbit antibody (ALI3404; BIOSourCE) diluted to 1/5000 with TBS-T containing 3% BSA was used as thesecond antibody. The AGF band was not detected in the AGF homozygous KOmice, to confirm that AGF was deficient.

Example 2 Preparation of CAG-AGF Tg Mice

In this example, AGF transgenic mice (hereinafter referred to as CAG-AGFTg mice) in which mouse AGF was systemically overexpressed under thecontrol of a CAG (modified chicken beta-actin promoter with CMV-IEenhancer) promoter [GENE, 108(1991) 193-200] were prepared. A plasmid inwhich the CAG promoter (1.7 kb), a lox71 sequence, a blasticidin gene(bsr), a poly A signal sequence (0.5 kb), a lox P sequence, a mouse AGFcDNA sequence, and an IRES (internal ribosomal entry site)-β-geo-poly Asequence (4.5 kb) were inserted at the multicloning site of plasmidpBluescriptII KS(+) (Stratagene) in this order was prepared inaccordance with the following procedures.

The full-length of mouse AGF cDNA prepared by the procedures describedin WO03/083114 was used as a template, together with a primer set [SEQID NO: 13 (AGAAGCTTCACCATGGGGACCGCCAGGCTAC; artificial sequence) and SEQID NO: 14 (CCGTCGACATTAGATCTTCACAAGCGCACAAGCCGGGTC; artificialsequence)] to carry out a PCR. In the PCR, a reaction at 95° C. for 10minutes was carried out, and a cycle composed of reactions at 94° C. for15 seconds, at 60° C. for 30 seconds, and at 72° C. for 2 minutes wasrepeated 45 times. The obtained PCR product was subcloned into a pZErO-2cloning vector (Invitrogen). The obtained plasmid was digested withrestriction enzymes HindIII and SalI, and inserted between the HindIIIand SalI sites of plasmid pBluescriptII SK (Stratagene) to constructplasmid pBS-mAGF containing the full-length of mouse AGF gene. Tointroduce the IRES-β-geo-poly A gene into the plasmid pBS-mAGF, plasmidpU-San (Hum Mol Genet 8:387-396 1999) carrying the IRES-β-geo-poly Agene was digested with restriction enzymes SalI and BglII, and theobtained IRES-β-geo-poly A gene was inserted between the BglII and SalIsites of pBS-mAGF to construct plasmid pBS-mAGF-βgeo containing themouse AGF cDNA sequence and the IRES-β-geo-poly A sequence.

A plasmid in which bsr and the poly A signal sequence are interposedbetween the lox71 sequence and the loxP sequence by inserting the loxPsequence into the 3′ side of the poly A signal of plasmid pCAGlox71bsr[Nucleic Acids Res. 1997; 25(4): 868-872] carrying the CAG promoter, thelox71 sequence, bsr, and the poly A signal sequence, was constructed asdescribed below. That is, a phosphorylated fragment of 81 bp (SEQ ID NO:15; GATCCGGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCCCTCGACCTGCAGCCCGGGGGATC; artificial sequence) carrying loxP was insertedinto the plasmid pCAGlox71bsr, which had been previously digested withrestriction enzyme SmaI and had been treated with BAP (bacterialalkaline phosphatase), to construct plasmid loxP-lox71. To confirm theloxP sequence inserted in the plasmid loxP-lox71, a nucleotide sequencecontaining the inserted loxP sequence was sequenced using a T3 primer(SEQ ID NO: 16; AATTAACCCTCACTAAAGGG). As a result, the nucleotidesequence determined by the T3 primer accorded with that of SEQ ID NO:15, and thus, it was confirmed that the loxP and lox71 sequences havethe same direction. The plasmid loxP-lox71 was digested with restrictionenzyme SpeI, and the obtained fragment containing the CAG promoter, thelox71 sequence, bsr, the poly A signal sequence, and the loxP sequencewas inserted at the SpeI site of plasmid pBS-mAGF-βgeo to construct thedesired plasmid pBS-loxP-lox71-mAGF-βgeo. The structure of the plasmidpBS-loxP-lox71-mAGF-βgeo is shown in FIG. 1. The plasmidpBS-loxP-lox71-mAGF-βgeo was digested with restriction enzyme NotI toobtain a linearized DNA fragment containing the CAG promoter, the lox71sequence, bsr, the poly A signal sequence, the loxP sequence, the mouseAGF cDNA sequence, and the IRES-β-geo-poly A sequence (pA).

The linearized DNA fragment was introduced into TT2 ES cells [Anal.Biochem., 1993, 214(1): 70-76] by electroporation (0.8 V, 3 μF). The EScells were cultured in the presence of 4 μg/mL blasticidin, and cells inwhich the genes were introduced were selected to establish 20 clones. ACAG-Cre vector (Blood, 1326-1333, Vol. 100, 2002) as a circular plasmidwas introduced into each clone by electroporation (0.8 V, 3 μF).

When Cre recombinase expressed by the CAG promoter excises the genebetween lox71 and loxP, AGF and β-geo will be expressed by the CAGpromoter activity. To select clones expressing β-geo, the ES cells werecultured in the presence of G418 (200 μg/mL) to select cells having thegenetic structure in which the region between lox71 and loxP weredeleted. ES cells (15 clones) selected at this stage have the CAGpromoter (1.7 kb), the lox71 sequence, the mouse AGF cDNA sequence, andthe IRES-β-geo-poly A sequence (4.5 kb), and express AGF constitutivelyunder the control of the CAG promoter. Each original clone beforeintroducing the CAG-Cre vector (i.e., ES cells in which the regionbetween lox71 and loxP was not deleted) was used as a negative controlto confirm that AGF was expressed in the selected 15 clone by Westernblotting using the anti AGF antibody described in Example 1(3). That is,ES cells were lysed with the lysis buffer, followed by an equal volumeof the 2×SDS sample buffer, the obtained samples were subjected toWestern blotting, and the expression of AGF was confirmed. From the EScells expressing AGF, three lines (8-1,8-2, and 9-1) were selected. Theselected cell lines were microinjected into blastocysts, and themanipulated eggs were transferred to a uterus to obtain chimeric micefrom the pregnant mice. The chimeric mice were mated with C57BL/6 miceto obtain transgenic mice expressing AGF constitutively under thecontrol of the CAG promoter. To identify the transgenic mice, a PCRusing genomic DNA isolated from the tail of each offspring mouse as atemplate was carried out, as described below. That is, the tail wastreated with proteinase K, and a phenol/chloroform extraction wascarried out to obtain DNA. The extracted DNA was collected by anisopropanol precipitation followed by an ethanol precipitation, anddissolved in a TE solution.

The following primers were designed on the basis of the mouse AGF cDNAsequence and a LacZ sequence: (AGF) Forward primer: SEQ ID NO: 17(5′-cccactacgacagcttctcc-3′; mouse) Reverse primer: SEQ ID NO: 18(5′-agccgggtcaacataacagc-3′; mouse) (LacZ) Forward primer: SEQ ID NO: 19(5′-gcgttacccaacttaatcg-3′; artificial sequence) Reverse primer: SEQ IDNO: 20 (5′-tgtgagcgagtaacaacc-3′; artificial sequence)

In PCRs using these primers, fragments of 325 bp and 320 bp areamplified from the introduced gene in PCRs for detecting the AGF cDNAand for detecting LacZ, respectively, and such fragments are notamplified from the mouse genomic DNA. The above primers and each genomicDNA prepared from the offspring mice were used to perform PCRs using aDNA polymerase (ExTaq; Takara). In the PCRs, a thermal denature at 94°C. for 5 minutes was carried out, a cycle composed of reactions at 94°C. for a minute, at 62° C. for a minute and 30 seconds, and at 72° C.for a minute and 30 seconds was repeated 28 times, and an elongationreaction at 72° C. for 7 minutes was carried out. The sizes of fragmentsamplified by the PCRs were analyzed. As a result, the expected bandswere detected in both PCRs for detecting the AGF cDNA and LacZ, withrespect to the three lines. The result shows that a germ linetransmission occurred in the three lines, and that the three lines aretransgenic mice expressing AGF constitutively under the control of theCAG promoter.

Example 3 Expression of AGF Gene in CAG-AGF Tg Mouse

In this example, the degree of the AGF gene expressed in the CAG-AGF Tgmouse prepared in Example 2 was analyzed. Total RNAs were prepared fromthe CAG-AGF Tg mouse and the littermate WT mouse [white adipose tissue(WAT), brown adipose tissue (BAT), cerebrum, cerebellum, hypothalamus,heart, liver, kidney, spleen, skeletal muscle, and pancreas] using atrizol reagent (Invitrogen). An commercially available RNA purificationreagent (RNeasy; Qiagen) and DNase (Qiagen) were used to perform a DNasetreatment and cleanup of the total RNAs. After the DNase treatment, 0.5μg of the total RNAs were converted to cDNAs using superscriptfirst-strand system for RT-PCR (LIFE TECHNOLOGIES).

Amounts of AGF and 18S ribosomal RNA (18SrRNA) expressed were determinedby a quantitative PCR method. The 18SrRNA was used as an internalstandard. The quantitative PCR was carried out by measuring an amount ofreal-time fluorescence using a sequence detection system (ABI PRISM7900HT Sequence Detection System; Applied Biosystems). The above cDNAswere used as a template, and the following primers and a Taq Man probedesigned for each gene were used as primers. Primers [SEQ ID NO: 21(TCGTGTAGTAGCCGTGTGGTGT; mouse) and SEQ ID NO: 22 (CACCTGATGCACAGGTTCCA;mouse)] and a commercially available PCR reagent (SYBR Green PCR MasterMix; Applied Biosystems) were used to carry out a PCR for measuring anamount of the AGF gene expressed. Primers [SEQ ID NO: 23(TGGTTGATCCTGCCAGTAG; mouse) and SEQ ID NO: 24 (CGACCAAAGGAACCATAACT;mouse)], a Taq Man probe [SEQ ID NO: 25 (CCGGTACAGTGAAACTGCGAATG;mouse)], and a commercially available PCR reagent (TaqMan Universal PCRMaster Mix; Applied Biosystems) were used to carry out a PCR formeasuring an amount of the 18SrRNA expressed.

In the PCR, an initial denaturing reaction at 95° C. for 10 minutes wascarried out, and a cycle composed of reactions at 94° C. for 15 secondsand at 60° C. for 60 seconds was repeated 45 times. Standard curves forcalculating amounts of genes expressed were prepared by using the abovecDNAs or mouse genomic DNA as a template. Amounts of genes expressedwere calculated as relative values between samples.

As a result, it was revealed that an amount of the AGF gene expressed intissues of the CAG-AGF Tg mouse was increased in comparison with that intissues of the WT mouse. In particular, increased expressions wereremarkably observed in skeletal muscle, BAT, and heart.

Example 4 Changes in Body Weight of Genetically Modified Mice

(1) Changes in Body Weight of CAG-AGF Tg Mice

Two generations of backcrosses of the CAG-AGF Tg mouse with C57BL/6 werecarried out to obtain CAG-AGF Tg mice (F2). The CAG-AGF Tg mice and thelittermate WT mice were normally bred with a normal diet (CE-2; CLEAJapan). When the body weights of 6-week-old female mice were measuredand compared, those of the CAG-AGF Tg mice and the WT mice were 15.7g±0.8 g (SD) and 17.8 g±0.5 g (SD), respectively. When those of12-week-old female mice were measured and compared, those of the CAG-AGFTg mice and the WT mice were 19.5 g±0.7 g (SD) and 23.5 g±2.5 g (SD),respectively. It was found from the results that the body weight of theCAG-AGF Tg mouse was lighter than that of the WT mouse.

Changes in body weight when the above mice were bred with a high fatdiet were examined. The mice were normally bred with a high fat diet(HFD-32; CLEA Japan) for 12 weeks, and changes in body weight wereanalyzed. As a result, the amounts of body weights increase for 12 weekswere 7.1 g±1 g (SD) in CAG-AGF Tg mice and 21.8 g±4 g (SD) in WT mice,respectively. The results show that when the CAG-AGF Tg mice were bredwith a normal diet, an increase in body weight was suppressed, and thatwhen the CAG-AGF Tg mice were bred with a high fat diet, an increase inbody weight was remarkably suppressed. It was found from the resultsthat AGF exhibits an activity of suppressing an increase in body weight.

(2) Changes in Body Weight of AGF KO Mice

The AGF homozygous KO mice, the AGF heterozygous KO mice, and thelittermate WT mice were normally bred with a normal diet. Each bodyweight was measured every week until 24-week-old. The results are shownin FIG. 2. As shown in FIG. 2, the body weight of the AGF homozygous KOmice was higher than that of the WT mice from approximately 12-week-old,and an increase in body weight of the AGF homozygous KO mice continuedto become remarkable obese. The AGF heterozygous mice exhibited anintermediate phenotype between those of the AGF homozygous KO mice andthe littermate WT mice.

Example 5 Changes in Organ Weight of Genetically Modified Mice

(1) Changes in Organ Weight in CAG-AGF Tg Mice

As described above, it was found that an increase in body weight wassuppressed in the CAG-AGF Tg mouse. In this example, the weights ofvarious organs were measured to reveal the mechanism. Each organ[genital fat pads (WAT), brown adipose tissue (BAT), liver, heart,kidney, and spleen] was obtained from the CAG-AGF Tg mice and thelittermate WT mice, and the weight of each organ per body weight wasmeasured. The measurement was carried out using 12-week-old mice bredwith a normal diet and mice bred with a high fat diet for 12 weeks (from12 week-old to 24-week-old). As a result, no changes were observed inBAT, liver, heart, kidney, and spleen between the mice (i.e., CAG-AGF Tgmice and WT mice) bred with a normal diet or a high fat diet. Incontrast, the weight per body weight of genital fat pads (white adiposetissue) in the CAG-AGF Tg mice bred with a normal diet or a high fatdiet was decreased in comparison with that in the WT mice. The resultsshow that, in the CAG-AGF Tg mouse, an increase in the weight of WAT wassuppressed, and thus, an increase in the body weight thereof wassuppressed. That is, it was found that AGF does not act on the weightsof organs other than WAT, but suppresses an increase in the weight ofadipose tissue accompanied by obesity. The results in WAT are shown inFIG. 3 (normal diet) and FIG. 4 (high fat diet), respectively.

(2) Changes in Organ Weight in AGF KO Mice

As described above, it was found that the body weight was increased inthe AGF KO mouse. In this example, the weights of various organs weremeasured to reveal the mechanism. Each

organ [genital fat pads (WAT), brown adipose tissue (BAT), liver, heart,kidney, and spleen] was obtained from 20-week-old female AGF homozygousKO mice, AGF heterozygous mice, and littermate WT mice bred with anormal diet, and the weight of each organ per body weight was measured.As a result, no changes were observed in BAT, liver, heart, kidney, andspleen among the AGF homozygous KO mice, the AGF heterozygous mice, andthe littermate WT mice. In contrast, the weight per body weight or permouse of genital fat pads in the AGF homozygous mice was increased incomparison with that in the WT mice. The AGF heterozygous mice exhibitedintermediate organ weights between those of the AGF homozygous KO miceand the WT mice. The results show that, in the AGF KO mouse, the weightof WAT such as genital fat pads was increased, and thus, the body weightthereof was increased. That is, it was found that the AGF KO mouseexhibits phenotypes opposite to the CAG-AGF Tg mouse, and that AGF doesnot act on the weights of organs other than WAT, but suppresses anincrease in the weight of adipose tissue. The results in genital fatpads (WAT) are shown in FIG. 5 (weight of WAT) and FIG. 6 (weight ofWAT/body weight), respectively.

Example 6 Changes in Form of Adipocytes in Genetically Modified Mice

(1) Changes in Form of Adipocytes in CAG-AGF Tg Mice

It is known that a high fat diet increases the weight of WAT andhypertrophy of adipocytes. It is known that the hypertrophy ofadipocytes is involved in deteriorating diabetes [IGAKU NO AYUMI, 192,513-518, 2000; and IGAKU NO AYUMI, 192, 541-545, 2000]. Therefore, theforms of adipocytes in the CAG-AGF Tg mice were analyzed in thisexample, as described below. Each fat tissue was obtained from CAG-AGFTg mice and littermate WT mice bred with a high fat diet for 12 weeksfrom 12-week-old to 24-week-old. Each tissue was fixed with a 10%formalin neutral buffer solution (Wako) and embedded in paraffin. Slicedsections were prepared and a hematoxylin and eosin (H&E) stain wascarried out. The result is shown in FIG. 7. In the littermate WT mouse(NTG), adipocytes became hypertrophied. In the CAG-AGF Tg mouse (TG),the hypertrophy of adipocytes was suppressed and the sizes thereof weremaintained as a normal size.

(2) Changes in Form of Adipocytes in AGF KO Mice

It is known that adipocytes become hypertrophied in model mice fordiabetes or obesity, accompanied by an increase in the weight ofadipocytes [Diabetologia, 14(3), 141-148, 1978]. It is known that thehypertrophy of adipocytes is involved in deteriorating diabetes, andthus, the forms of adipocytes in the AGF KO mice were analyzed in thisexample, as described below. Each genital fat pads (WAT) and brown fattissue (BAT) were obtained from the AGF homozygous mice and thelittermate WT mice. Each tissue was fixed with a 10% formalin neutralbuffer solution and embedded in paraffin. Sliced sections were preparedand a hematoxylin and eosin (H&E) stain was carried out. The results areshown in FIG. 8 (AGF homozygous KO mouse) and FIG. 9 (littermate WTmouse), respectively. It was found that adipocytes in WAT of thelittermate WT mice had a normal size, and that adipocytes in WAT of theAGF homozygous KO mice became hypertrophied. Further, an accumulation offat in BAT of the AGF homozygous KO mice was observed in comparison withthat of the littermate WT mice.

Example 7 Changes in Triglyceride Content in Tissues of GeneticallyModified Mice

(1) Changes in Triglyceride Content in Tissues of CAG-AGF Tg Mice

It is known that obesity causes not only an increase in fat tissues, butalso an increase in triglyceride (TG) content in skeletal muscles orliver (Nippon Rinsho, 1995, vol. 53, Special Issue in 1995, Himansho, p.354-p358). In this example, TG contents in skeletal muscles(gastrocnemial muscles) and liver of the CAG-AGF Tg mice were analyzed,as described below. The CAG-AGF Tg mice (Tg) and the littermate WT mice(WT) were bred with a high fat diet (HFD-32; CLEA Japan) for a month orthree months. A chloroform-methanol solution was used to extract TG fromskeletal muscles and liver of each mouse (seikagakujikkenkouza 3,shishitsunokagaku, tokyo kagaku doujin). A concentration of eachextracted TG was measured using a kit (Triglyceride E test Wako; Wako)to determine TG contents in the tissues. The results are shown in FIG.10 (liver) and FIG. 11 (skeletal muscles), respectively. It was foundthat each TG content in tissues (skeletal muscles or liver) of theCAG-AGF Tg mice was decreased in a comparison with that in the WT mice.It was found that AGF exhibits an activity of decreasing TG contents inskeletal muscles or liver. In this connection, it is known that the TGcontents in skeletal muscles or liver are increased by obesity.

(2) TG Contents in Tissues of AGF KO Mice

TG contents in skeletal muscles (gastrocnemial muscles) and liver of theAGF KO mice were analyzed. The method described in Example 7(1) wasrepeated, except for that the AGF KO mice and the littermate WT micewere used, to extract TG therefrom. A concentration of each extracted TGwas measured using a kit (Triglyceride E test Wako; Wako) to determineTG contents in the tissues. The results are shown in FIG. 12. It wasfound that TG contents in skeletal muscles and liver of the AGF KO micewere remarkably increased in comparison of that in the WT mice. It wasfound that the AGF KO mouse exhibits phenotypes opposite to the CAG-AGFTg mouse, and that AGF exhibits an activity of decreasing TG contents inskeletal muscles or liver.

Example 8 Changes in Blood Glucose Level and Blood Insulin Concentrationin AGF KO Mice

A glucose tolerance test for the AGF homozygous KO mice and thelittermate WT mice was carried out to analyze a blood glucose level anda concentration of blood insulin, as described below. The mice were madeto fast for 16 hours, and 1 g/kg of D-glucose was intraperitoneallyadministered. Blood was taken from ophthalmic veins before theadministration, and at 15, 30, 60, and 120 minutes after theadministration. The blood glucose level was measured using GLUTEST ACE(SANWA KAGAKU KENKYUSHO), and the concentration of blood insulin wasmeasured using a RIA2 antibody method (SRL). The results are shown inFIG. 13 (blood glucose value) and FIG. 14 (insulin in sera),respectively.

In the WT mice, the blood glucose level increased by the glucoseadministration began to decrease at 30 minutes after the administration.In the AGF homozygous mice, the blood glucose level was greatlyincreased by the glucose administration, and the elevated blood glucosevalue did not begin to decrease at 60 minutes after the administration.It was found from the results that the AGF homozygous KO mice exhibitedan abnormality in glucose tolerance. With respect to the concentrationin blood insulin, it was increased by the glucose administration in theWT mice, whereas the concentration of blood insulin in the AGFhomozygous KO mice was remarkably high before the glucoseadministration. This shows that the AGF homozygous KO mice suffered fromhyperinsulinemia. From the results, it was found that the AGF homozygousKO mouse deficient in the AGF gene suffered from diabetes, and that AGFexhibits an antidiabetic activity.

Example 9 Oxygen Consumption in CAG-AGF Tg Mice

The oxygen consumption in the CAG-AGF Tg mice and the littermate WT micewas measured in this example. An apparatus for measuring oxygenconsumption (OXYMAX; Columbus Instruments) was used to measure oxygenconsumption in fasting mice for 24 hours in accordance with a manualattached to the apparatus. Oxygen consumption in 14 CAG-AGF Tg mice and14 littermate WT mice was measured to determine and prepare oxygenconsumption for a 12-hour light period (7:00˜19:00), that for a 12-hourdark period (19:00˜7:00), and that for 24 hours. The results are shownin FIG. 15. The oxygen consumption (VO₂) in the CAG-AGF Tg mice washigher than that in the WT mice in any time zone. It was found that AGFexhibits an activity for promoting oxygen consumption. Because oxygenconsumption correlates with energy consumption, the promotion of oxygenconsumption exhibits an antiobesity activity [FEBS Letters, 491(1-2):154-158, 2001]. It was found that AGF exhibits an activity of promotingoxygen consumption. The result supports the antiobesity activity of AGF.

Example 10 Changes in Genes Expressed in Tissues of CAG-AGF Tg Mice

In this example, changes in a UCP (Uncoupling Protein) gene and a PPAR(Peroxisome Proliferator-Activated Receptor) gene expressed in brownadipose tissue (BAT) and skeletal muscles of the CAG-AGF Tg mice wereanalyzed. In accordance with the procedures described in Example 3,total RNAs were prepared from BAT and skeletal muscles of the CAG-AGF Tgmice and the littermate WT mice, and were treated with DNase, and cDNAswere synthesized. Amounts of UCP1, UCP3, PPAR-α, PPAR-δ, and β-actinexpressed were determined by the quantitative PCR method, described inExample 3. Primers shown in Table 1 were used in the quantitative PCR.Further, as commercially available PCR reagents, SYBR Green PCR MasterMix (Applied Biosystems) was used for β-actin, and TaqMan Universal PCRMaster Mix (Applied Biosystems) was used for UCP1, UCP3, PPAR-α, andPPAR-5. TABLE 1 Genes Forward primer Reverse primer TaqMan primer UCP 1SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 UCP 3 SEQ ID NO: 29 SEQ ID NO:30 SEQ ID NO: 31 PPAR-α SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 PPAR-δSEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 β-actin SEQ ID NO: 38 SEQ IDNO: 39 Not used

As a result, it was found that the expression of UCP1 was induced in BATof the CAG-AGF Tg mice, and that the expressions of UCP3, PPAR-α, andPPAR-δ were induced in skeletal muscles of the CAG-AGF Tg mice. That is,it was found that AGF induces the expression of UCP1 in BAT and those ofUCP3, PPAR-α, and PPAR-δ in skeletal muscles. Accordingly, it wasrevealed that AGF promotion in expression of UCP promoting heatconsumption and in expression of PPAR promoting heat consumption orlipid metabolism is one of the mechanisms of AGF activities forpromoting oxygen consumption, suppressing an increase in body weight,and suppressing an increase in the weight of adipose tissues.

Referential Example 1 Expression and Purification of Mouse AGF and HumanAGF

The human AGF and the mouse AGF were expressed and purified inaccordance with the procedures described in WO03/083114 (Example 19) asdescribed below. That is, DNA fragments of approximately 1.4 kbp (human)and approximately 1.3 kbp (mouse) were independently inserted intoplasmid pcDNA-Signal-FLAG. Each resulting expression plasmid wasintroduced into HEK293 cells. Each culture supernatant of the cellsexpressing the human AGF or the mouse AGF was purified by affinitychromatograph using anti FLAG-M2 monoclonal antibody agarose affinitygel (Sigma) to obtain human and mouse recombinant AGF proteins.

Referential Example 2 Changes in Genes Expressed in C2C12 CellsDifferentiated into Skeletal Muscle by AGF Stimulation

After C2C12 cells (obtained from ATCC) were cultured to the confluentconditions, the culture medium was changed from DMEM with 10% fetal calfserum (FCS) to DMEM with 2.5% horse serum, and the cultivation wasfurther continued for 8 days to differentiate the cells to skeletalmuscle cells. After starvation of C2C12 cells differentiated intoskeletal muscle for 12 hours, 3 ug/mL of human or mouse AGF recombinantprotein were added or not added to the cells, and the cells were allowedto stand for 4, 8, 12, and 24 hours thereafter. Total RNAs were preparedfrom AGF-stimulated cells and nonstimulated cells, and treated withDNase, and cDNAs were synthesized. Amounts of PPAR-α, PGC-1α (PeroxisomeProliferator-Activated Receptor-coactivator 1 alpha), and CYP(cyclophilin) genes expressed were measured by the quantitative PCRdescribed in Example 10. Primers listed in Tables 1 and 2 were used inthe quantitative PCR. As commercially available PCR reagents, SYBR GreenPCR Master Mix (Applied Biosystems) was used for CYP, and TaqManUniversal PCR Master Mix (Applied Biosystems) was used for PPAR-A andPGC-1α. An amount of a LCAD (long-chain acyl-CoA dehydrogenase) geneexpressed was measured by using a commercially available assay system(Assay on demand; Applied Biosystems) and TaqMan Universal PCR MasterMix (Applied Biosystems) (Assay ID: Mm00599660_m1). The proceduresdescribed in Example 10 were repeated, except that reagents contained inthe above purchased assay system were used as primers and PCR reactionsolutions, in accordance with attached protocols. TABLE 2 Genes Forwardprimer Reverse primer TaqMan primer PGC-1α SEQ ID NO: 40 SEQ ID NO: 41SEQ ID NO: 42 CYP SEQ ID NO: 43 SEQ ID NO: 44 Not used

As a result, it was found that the stimulation of the human or mouse AGFpromotes expressions of the PPAR-α, PGC-1α, and LCAD genes. It showsthat AGF promotes heat consumption and fatty acid oxidation in skeletalmuscle cells. It is known that PPAR-α or PGC-1α promotes lipidmetabolism or heat consumption [Acta Physiologica Scandinavica.178(4):425-434, 2003; and Endocrine Reviews 24(1):78-90, 2003]. AGFpromoted the expressions of PPAR-α, LCAD, and PGC-1α, and thus, theresult supports the antiobesity activity of AGF.

Example 11 Cloning of Upstream Sequence of hAGF mRNA TranscriptionalRegion

(1) Cloning of Sequences of Approximately 200, 300, 400, 600, and 800 bpUpstream of the hAGF mRNA Transcriptional Region

Human genome (Genomic DNA; Clontech) was used as a template to carry outPCR with DNA polymerase (TaKaRa LA taq™; Takara). In the PCR, a reactionat 95° C. for 2 minutes was carried out, a cycle composed of reactionsat 94° C. for 30 seconds, at 60° C. for 30 seconds, and at 72° C. for aminute was repeated 40 times, and a reaction at 72° C. for 5 minutes wascarried out. As primer sets, five primer sets (SEQ ID NOS: 45 and 46,SEQ ID NOS: 47 and 46, SEQ ID NOS: 48 and 46, SEQ ID NOS: 49 and 46, andSEQ ID NOS: 50 and 46) were used. Fragments of approximately 200, 300,400, 600, and 800 bp obtained by using the above primer sets wereindependently subcloned into a cloning vector (pCR2.1-TOPO; Invitrogen).Each subclone was digested with restriction enzymes KpnI and NheI, andthe obtained fragments (nucleotides 2790-3001, 2705-3001, 2604-3001,2406-3001, and 2206-3001 of SEQ ID NO: 1) were independently insertedinto vector pGV-B2 for a luciferase assay system (PicaGene Vector 2basic vector; Toyo Ink) previously digested with KpnI and NheI, toobtain pGV-hAGFpro200(N6), pGV-hAGFpro300(N6), pGV-hAGFpro400(N6),pGV-hAGFpro600(N6), and pGV-hAGFpro800(N6).

As described above, nucleotide sequences consisting of nucleotides2790-3001, 2705-3001, 2604-3001, 2406-3001, and 2206-3001 of SEQ ID NO:1, i.e., regions of approximately 200, 300, 400, 600, and 800 bpupstream of a human AGF mRNA transcriptional region, were cloned, andplasmids capable of measuring promoter activities thereof wereconstructed.

(2) Cloning of Sequences of Approximately 1, 1.3, and 3 kbp Upstream ofthe hAGF mRNA Transcriptional Region

Cloning of sequences of approximately 1 kbp and 1.3 kbp upstream of thehAGF mRNA transcriptional region was carried out by PCR, as describedbelow. A forward primer consisting of the nucleotide sequence of SEQ IDNO: 51 and a reverse primer consisting of the nucleotide sequence of SEQID NO: 52 were used for cloning the region of approximately 1 kb. Aforward primer consisting of the nucleotide sequence of SEQ ID NO: 53and a reverse primer consisting of the nucleotide sequence of SEQ ID NO:52 were used for cloning the region of approximately 1.3 kb. Humangenome (Genomic DNA; Clontech) was used as a template to carry out PCRwith DNA polymerase (TaKaRa LA taq™; Takara) and the above primer sets.In the PCR, a reaction at 95° C. for 2 minutes was carried out, a cyclecomposed of reactions at 94° C. for 30 seconds, at 60° C. for 30seconds, and at 72° C. for 3 minutes was repeated 45 times, and areaction at 72° C. for 5 minutes was carried out. Obtained fragments ofapproximately 1 kbp and 1.3 kbp were independently subcloned into acloning vector (pCR-XL-TOPO; Invitrogen). Each subclone was digestedwith restriction enzymes KpnI and NheI, and the obtained fragments(nucleotides 2021-3028 and 1640-3028 of SEQ ID NO: 1) were independentlyinserted into vector pGV-B2 previously digested with KpnI and NheI, toobtain pGV-hAGFpro1k(N4) and pGV-hAGFpro1.3k(N4).

Further, human genome (Genomic DNA; Clontech) was used as a template tocarry out PCR with DNA polymerase (TaKaRa LA taq™; Takara) and a primerset of a forward primer consisting of the nucleotide sequence of SEQ IDNO: 54 and a reverse primer consisting of the nucleotide sequence of SEQID NO: 55. In the PCR, a reaction at 95° C. for 2 minutes was carriedout, a cycle composed of reactions at 94° C. for 30 seconds, at 63° C.for 30 seconds, and at 72° C. for 2 minutes and 30 seconds was repeated45 times, and a reaction at 72° C. for 5 minutes was carried out

The resulting fragment of approximately 2 kb was subcloned into acloning vector (pCR-XL-TOPO; Invitrogen). The obtained subclone wasdigested with KpnI and XmaI to obtain a fragment containing thenucleotide sequence consisting of nucleotides 1-1768 of SEQ ID NO: 1.The pGV-hAGFpro1.3k(N4) plasmid contains two XmaI recognition sites,i.e., a XmaI recognition site in the sequence upstream of the hAGF mRNAtranscriptional region and a XmaI recognition site in the multicloningsite of the pGV-B2 vector. Therefore, 8.7 μg of pGV-hAGFpro1.3k(N4) wasdigested with 5 units of XmaI for 1 to 10 minutes to prepare a mixtureof plasmids cleaved at 0, 1, or 2 sites. The plasmid cleaved at only onesite was separated and extracted by electrophoresis. The obtainedplasmid was digested with KpnI, and a fragment of 6.1 kbp (determined byelectrophoresis) was taken to obtain a plasmid in which the XmaI site inthe cloning site of pGV-B2 was not cleaved and the XmaI site in the hAGFpromoter region was cleaved. To the plasmid, the fragment containing thenucleotide sequence consisting of nucleotides 1-1768 of SEQ ID NO:1,obtained by the above-described procedure (i.e., treatment with KpnI andXmaI), was inserted to obtain pGV-hAGFpro3k(N4).

The plasmid pGV-hAGFpro800(N6) was digested with SnaBI and XbaI toobtain a fragment (N6 fragment) of approximately 2 kbp. The plasmidspGV-hAGFpro1k(N2), pGV-hAGFpro1.3k(N4), and pGV-hAGFpro3k(N4) weredigested with SnaBI and XbaI to remove the excised fragment ofapproximately 2 kbp. The N6 fragment was inserted thereinto to obtainpGV-hAGFhAGFpro1k(N6), pGV-hAGFpro1.3k(N6), and pGV-hAGFpro3k(N6). Theseobtained plasmids contain a hAGF promoter region having nucleotidesequence consisting of nucleotides 2021-3001, 1640-3001, or 1-3001 ofSEQ ID NO: 1 in the reporter plasmid.

As described above, nucleotide sequences consisting of nucleotides2021-3001, 1640-3001, and 1-3001 of SEQ ID NO: 1, i.e., regions ofapproximately 1 kbp, 1.3 kbp, and 3 kbp upstream of the human AGF mRNAtranscriptional region, were cloned, and plasmids capable of measuringpromoter activities thereof were constructed.

Example 12 Analysis of DNA Sequence of Human AGF Promoter Region

293EBNA cells (Invitrogen) cultured in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal calf serum, 100 μg/mLpenicillin, and 100 μg/mL streptomycin were transfected with i) pGV-B2(control vector), or plasmids obtained in Example 11 [i.e.,pGV-hAGFpro200(N6), pGV-hAGFpro300(N6), pGV-hAGFpro400(N6),pGV-hAGFpro600(N6), pGV-hAGFpro800(N6), pGV-hAGFpro1k(N6),pGV-hAGFpro1.3k(N6), or pGV-hAGFpro3k(N6)] and ii) a β-gal expressingplasmid (pCH110; Amersham pharmacia biotech), using a transfectionreagent (FuGene-6; Nippon Roche). A luciferase activity after 48-hourcultivation under conventional culture conditions was measured by acommercially available measuring kit (PicaGene. luminescence kit; ToyoInk). The measuring values were compensated on the basis of activitiesof β-gal expressed by the co-transfected β-gal expressing plasmid. Theβ-gal activity was measured by a commercially available measuring kit(Galacto-Light Plus kit; Roche). As a result, no increased luciferaseactivities were observed in cells transfected with plasmids containingDNAs of approximately 400 bp to 3 kbp [pGV-hAGFpro400(N6),pGV-hAGFpro600(N6), pGV-hAGFpro800(N6), pGV-hAGFpro1k(N6),pGV-hAGFpro1.3k(N6), or pGV-hAGFpro3k(N6)]. Unexpectedly, a remarkablyincreased luciferase activity, which was not observed in cellstransfected with the control vector, was observed in cells transfectedwith the plasmid pGV-hAGFpro300(N6) containing the shorter DNA ofapproximately 300 bp (FIG. 16). No increased luciferase activity wasobserved in cells transfected with the plasmid pGV-hAGFpro200(N6)containing the further shorter DNA of approximately 200 bp (FIG. 17).The results show that a promoter activity is located in the region ofapproximately 300 bp consisting of the nucleotide sequence 2705-3001 ofSEQ ID NO: 1, and that a silencer sequence capable of suppressing thepromoter activity is located in the sequence upstream of the abovepromoter region of approximately 300 bp.

The present inventors constructed a reporter assay utilizing the DNAsequence of approximately 300 bp consisting of the nucleotide sequenceconsisting of nucleotides 2705-3001 of SEQ ID NO: 1, and found that theregion contains the AGF promoter activity. An antiobesity agent, anantidiabetic agent, and/or a hypolipidemic agent, which promote thepromoter activity of the AGF gene and promote the expression of AGF, maybe screened by the assay system, preferably by bringing a test compoundinto contact with cells transfected with pGV-hAGFpro300(N6) andanalyzing the change in the luciferase activity.

Referential Example 3 Cloning of Mouse AGF

The mouse AGF was amplified by using a forward primer consisting of thenucleotide sequence of SEQ ID NO: 56 and a reverse primer consisting ofthe nucleotide sequence of SEQ ID NO: 57. In the PCR, Pyrobest DNApolymerase (Takara) was used, and a cycle consisting of reactions at 98°C. for 20 seconds, at 64° C. for 30 seconds, and at 74° C. for 3 minuteswas repeated 35 times in the presence of 5% formamide, to amplify a DNAfragment of approximately 1.5 kbp. The fragment was cloned into a pCR2.1plasmid (Invitrogen) to obtain plasmid pCR2.1-mNew. The nucleotidesequences of the obtained clones were analyzed by a DNA sequencer(ABI377 DNA Sequencer; Applied Biosystems) utilizing adideoxytermination method. The determined sequence is shown as SEQ IDNO: 58. The sequence contains an open reading frame consisting of 1374nucleotides (nucleotides 1-1374 of SEQ ID NO: 58). The amino acidsequence (457 amino acids) deduced from the open reading frame is shownas SEQ ID NO: 59. The mouse AGF contains a signal sequence (−24 to −1)at the N-terminus. The signal sequence is cleaved when AGF is secretedto the outside of cells. Mouse matured AGF consisting of the amino acidsequence consisting of amino acids 1-433 of SEQ ID NO: 59 after cleavingthe signal sequence exhibits physiological activities.

The sequence (mouse) consisting of the amino acid sequence consisting ofamino acids 1-433 of SEQ ID NO: 59 has a 76% homology with that (aminoacids 21-470 in Genbank accession No. NP_(—)114123) of human AGF knownas human NL8/NEW, angiopoietin-like 6, or angiopoietin-related protein5. In particular, a fibrinogen domain at the C-terminal side has a highhomology (89%). In this connection, it is known that the fibrinogendomain plays an important role in the activities of proteins belongingto an angiopoietin family [William N. Procopio et al., J. Biol. Chem.274: 30196-30201 (1999)]. From such a high homology in the region whichis considered to be important in maintaining the activities, it isconsidered that the mouse AGF of SEQ ID NO: 58 is a counterpart of thehuman AGF and that the mouse AGF and the human AGF have the sameactivities. In this connection, the homologies between amino acidsequences were calculated by a BLAST search algorithm. Moreparticularly, it may be calculated using a b12seq program (Tatiana A.Tatusova and Thomas L. Madden, FEMS Microbiol. Lett., 174, 247-250,1999) in a BLAST package (sgi32 bit edition, version 2.0.12; obtainedfrom NCBI) in accordance with a default parameter. As a pairwisealignment parameter, a program “blastp” is used. Further, “0” as a Gapinsertion cost value, “0” as a Gap elongation cost value, “SEG” as afilter for a Query sequence, and “BLOSUM62” as a Matrix are used,respectively.

Referential Example 4 Preparation of OP9 Cell Line Expressing AGF

Plasmid pCR2.1-mNew prepared in Referential example 3 was digested withrestriction enzymes XbaI and SpeI to obtain a fragment of 1.4 kbpcontaining the mouse AGF gene. The fragment was inserted intopEF-BOS-neo [Mizushima, S., & Nagata, S. Nucleic Acids Res. 18: 5322(1990)], which had been previously digested with XbaI and treated withBAP, to prepare an expression vector pEF-BOS-mAGF for expressing mouseAGF.

Fugene6 (Roche Diagnostics) was used in accordance with a protocolattached thereto to transfect OP9 cells [Nakano T., Semin. Immunol.7(3), 197-203, 1995] with pEF-BOS-mAGF. The transfected cells werecultured in the presence of 300 μg/mL Geneticin (Roche Diagnostics) toobtain a cell line (OP9/AGF) stably expressing mouse AGF. As a negativecontrol, an OP9 cell line (OP9/vector) transfected with the pEF-BOS-neovector without the mouse AGF gene was used.

Referential Example 5 Activity of AGF for Proliferating Chondrocytes

An activity of AGF for proliferating chondrocytes was analyzed in vitroas described below. Mouse chondrogenic cell line ATDC5 [Atsumi T. etal., Cell Differ. Dev. 30(2), 109-116, 1990] was infected with anexpression vector pEGFPMY [Onai N. et al., Blood, 96(6), 2074-2080,2000] for expressing green fluorescent protein (GFP) which may be usedin a retrovirus expression system. The preparation of retrovirus and theinfection to ATDC5 were carried out in accordance with the Miyamoto etal. method [Miyamoto T. et al., Blood, 98(8), 2544-2554, 2001].

The infected cells were cultured in an ATDC5 culture medium [DMEM/F-12(Lifetechnologies), 5% FCS, 5 μg/mL insulin, 5 μg/mL transferrin, and3×10⁻⁸ mol/L sodium selenite]. Before reaching confluent conditions,cells were treated with trypsin to detach them from the culture plate.The cells were suspended and subjected to a cell sorter (FACS vantage;Becton Dickinson). ATDC5 cells having fluorescence derived from GFP wereseparated and collected to obtain ATDC5 cells expressing GFP. Theobtained GFP-expressing ATDC5 cells were cultured and proliferated, andthe above procedures (i.e., separation and collection of GFP-expressingcells by the cell sorter) were repeated to obtain ATDC5 (ATDC5/GFP)stably expressing GFP. The AGF-stably-expressing OP9 cell line (OP9/AGF)and the control OP9 cell line (OP9/vector) prepared in Referentialexample 4 were cultured in a 12-well plate to become confluent. TheATDC5/GFP cells (50 cells/well) were added to each well of the plate,and cultured in the ATDC5 culture medium for 14 days. The proliferationof ATDC5/GFP was analyzed using a fluorescent microscope.

As a result, when the control OP9 cell line (OP9/vector) was used as afeeder cell, no ATDC5/GFP colony was formed, and ATDC5/GFP cells werenot proliferated. In contrast, when the AGF-stably-expressing OP9 cellline (OP9/AGF) was used as a feeder cell, many ATDC5/GFP colonies wereformed (approximately 16 colonies per well), and ATDC5/GFP cells wereremarkably proliferated. Because OP9/AGF expresses the recombinant AGFprotein, it was found that the recombinant AGF exhibits an activity ofproliferating ATDC5/GFP cells, i.e., the recombinant AGF exhibits anactivity of proliferating chondrocytes.

INDUSTRIAL APPLICABILITY

The AGF promoter, the screening method, and the nonhuman knockout animalaccording to the present invention may be used in screening anantiobesity agent, an antidiabetic agent, and/or a hypolipidemic agent.The nonhuman transgenic animal of the present invention may be used indeveloping an antiobesity agent, an antidiabetic agent, and/or ahypolipidemic agent.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[FIG. 1]

FIG. 1 is a schematic drawing showing the structure of plasmidpBS-loxP-lox71-mAGF-βgeo. The abbreviations “pro.” and “pri.” mean apromoter and a primer, respectively.

[FIG. 2]

FIG. 2 is a graph showing changes in body weight of AGF KO mice. Thehorizontal axis indicates an age in weeks (weeks), and the vertical axisindicates body weight (g). The abbreviations “WT”, “HTR”, and “HM” meanWT mice, heterozygous KO mice, and homozygous KO mice, respectively.

[FIG. 3]

FIG. 3 is a graph showing changes in the weight of genital fat pads(white adipose tissue) of CAG-AGF Tg mice (normal diet). The verticalaxis indicates [weight of white adipose tissue (g)]/[body weight (g)].The abbreviations “WT” and “Tg” mean WT mice and Tg mice, respectively.

[FIG. 4]

FIG. 4 is a graph showing changes in the weight of genital fat pads(white adipose tissue) of CAG-AGF Tg mice (high fat diet). The verticalaxis indicates [weight of white adipose tissue (g)]/[body weight (g)].The abbreviations “WT” and “Tg” mean WT mice and Tg mice, respectively.

[FIG. 5]

FIG. 5 is a graph showing changes in the weight of genital fat pads(white adipose tissue) of AGF KO mice (weight of white adipose tissue).The vertical axis indicates the weight of white adipose tissue (g). Theabbreviations “WT”, “HTR”, and “HM” mean WT mice, heterozygous KO mice,and homozygous KO mice, respectively.

[FIG. 6]

FIG. 6 is a graph showing changes in the weight of genital fat pads(white adipose tissue) of AGF KO mice (weight of white adiposetissue/body weight). The vertical axis indicates [weight of whiteadipose tissue (g)]/[body weight (g)]. The abbreviations “WT”, “HTR”,and “HM” mean WT mice, heterozygous KO mice, and homozygous KO mice,respectively.

[FIG. 7]

FIG. 7 is a microphotograph showing the form of adipocytes in theCAG-AGF Tg mouse.

[FIG. 8]

FIG. 8 is a microphotograph showing the form of adipocytes in the AGFhomozygous KO mouse.

[FIG. 9]

FIG. 9 is a microphotograph showing the form of adipocytes in thelittermate WT mouse.

[FIG. 10]

FIG. 10 is a graph showing TG contents in tissue (liver) of the CAG-AGFTg mouse. The horizontal axis indicates the period of breeding with ahigh fat diet (month). The vertical axis indicates a TG content (mg/gtissue). The abbreviations “WT” and “Tg” mean WT mice and Tg mice,respectively.

[FIG. 11]

FIG. 11 is a graph showing TG contents in tissue (skeletal muscles) ofthe CAG-AGF Tg mouse. The horizontal axis indicates the period ofbreeding with a high fat diet (months). The vertical axis indicates a TGcontent (mg/g tissue). The abbreviations “WT” and “Tg” mean WT mice andTg mice, respectively.

[FIG. 12]

FIG. 12 is a graph showing TG contents in tissue of the AGF KO mice. Thevertical axis indicates a TG content (mg/g tissue). The abbreviations“L” and “SM” in the horizontal axis mean the liver and skeletal muscles,respectively. The abbreviations “WT”, “HTR”, and “HM” mean WT mice,heterozygous KO mice, and homozygous KO mice, respectively.

[FIG. 13]

FIG. 13 is a graph showing the result (blood glucose level) of glucosetolerance test in the AGF KO mice. The horizontal axis indicates a time(minutes), and the vertical axis indicates a blood glucose level(mg/dL). The abbreviations “WT” and “HM” mean WT mice and homozygous KOmice, respectively.

[FIG. 14]

FIG. 14 is a graph showing the result (serum insulin) of glucosetolerance test in the AGF KO mice. The horizontal axis indicates a time(minutes), and the vertical axis indicates serum insulin (ng/mL). Theabbreviations “WT” and “HM” mean WT mice and homozygous KO mice,respectively.

[FIG. 15]

FIG. 15 is a graph showing oxygen consumption in the CAG-AGF Tg mice. Inthe horizontal axis, lane 1, lane 2, and lane 3 indicate results in thelight period, the dark period, and 24 hours, respectively. The verticalaxis indicates VO₂ (mL/kg/min). The abbreviations “WT” and “Tg” mean WTmice and Tg mice, respectively.

[FIG. 16]

FIG. 16 is a graph showing a luciferase activity. The vertical axisindicates luciferase/β-gal.

[FIG. 17]

FIG. 17 is a graph showing a luciferase activity. The vertical axisindicates luciferase/β-gal.

Free Text in Sequence Listing

Features of “Artificial Sequence” are described in the numericidentifier <223> in the Sequence Listing. More particularly, each of thenucleotide sequences of SEQ ID NOS: 6, 7, 9, 10, 13, 14, 16, 19, 20, 45to 53, and 56 is an artificially synthesized primer sequence. Thenucleotide sequence of SEQ ID NO: 15 is a sequence containing loxP.

1. (a) A DNA exhibiting a promoter activity for an angiopoietin-relatedgrowth factor, and consisting of a nucleotide sequence in which 1 to 10nucleotides are substituted, deleted, added, and/or inserted in thenucleotide sequence consisting of nucleotides 2705-3001 of SEQ ID NO: 1,or (b) a DNA exhibiting a promoter activity for an angiopoietin-relatedgrowth factor, and consisting of a nucleotide sequence having a 90% ormore homology with that consisting of nucleotides 2705-3001 of SEQ IDNO:
 1. 2. A DNA consisting of the nucleotide sequence consisting ofnucleotides 2705-3001 of SEQ ID NO:
 1. 3. A recombinant vectorcharacterized by comprising the DNA according to claim 1, and exhibitinga promoter activity for an angiopoietin-related growth factor.
 4. Atransformant characterized by comprising the DNA according to claim 1,and exhibiting a promoter activity for an angiopoietin-related growthfactor.
 5. A method for screening an antiobesity agent, an antidiabeticagent, and/or a hypolipidemic agent, characterized by comprising thesteps of: i) bringing a substance to be tested into contact with thetransformant according to claim 4, and ii) measuring a promoter activityfor an angiopoietin-related growth factor and analyzing a test substancedependent change in the promoter activity.
 6. The screening methodaccording to claim 5, wherein the transformant contains a reporter genelocated downstream of the DNA and the promoter activity for anangiopoietin-related growth factor is measured by analyzing anexpression of the reporter gene.
 7. A nonhuman knockout animalcharacterized in that a polynucleotide encoding an angiopoietin-relatedgrowth factor is functionally deficient on a chromosome.
 8. A nonhumantransgenic animal which is a nonhuman animal or an offspring animalthereof obtained by ontogenesis from totipotent cells in which apolynucleotide is introduced together with a CAG promoter, wherein thepolynucleotide is carried on a chromosome, a polypeptide encoded by thepolynucleotide is expressed in a somatic cell, and the polypeptide isselected from the group consisting of: (a) a polypeptide exhibiting anactivity of suppressing an increase in body weight, and comprising anamino acid sequence consisting of amino acids 1-450 of SEQ ID NO: 3 oramino acids 1-433 of SEQ ID NO: 5, (b) a polypeptide exhibiting anactivity of suppressing an increase in body weight, and comprising anamino acid sequence in which 1 to 10 amino acids are substituted,deleted, and/or inserted in an amino acid sequence consisting of aminoacids 1-450 of SEQ ID NO: 3 or amino acids 1-433 of SEQ ID NO: 5, (c) apolypeptide exhibiting an activity of suppressing an increase in bodyweight, and encoded by a DNA which hybridizes under stringent conditionsto a DNA encoding an amino acid sequence consisting of amino acids 1-450of SEQ ID NO: 3 or amino acids 1-433 of SEQ ID NO: 5, and (d) apolypeptide exhibiting an activity of suppressing an increase in bodyweight, and comprising an amino acid sequence having a 95% or morehomology with that consisting of amino acids 1-450 of SEQ ID NO: 3 oramino acids 1-433 of SEQ ID NO: 5.